Diamide compounds having muscarinic receptor antagonist and BETA2 adrenergic receptor agonist activity

ABSTRACT

This invention relates to a compound of formula I: 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof. Such compounds possess both muscarinic receptor antagonist and β 2  adrenergic receptor agonist activities. The invention also relates to pharmaceutical compositions comprising such compounds, processes and intermediates for preparing such compounds, and methods of using such compounds as bronchodilating agents to treat pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/327,853, filed on Jul. 10, 2014; which application is a divisional ofU.S. application Ser. No. 13/964,484, filed on Aug. 12, 2013 (now U.S.Pat. No. 8,816,088 B2); which application is a continuation of U.S.application Ser. No. 13/369,109, filed on Feb. 8, 2012 (now U.S. Pat.No. 8,551,978 B2); which application is a divisional of U.S. applicationSer. No. 12/761,532, filed on Apr. 16, 2010 (now U.S. Pat No. 8,138,345B2) which application claims the benefit of U.S. Provisional ApplicationNo. 61/172,039, filed on Apr. 23, 2009; the entire disclosures of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel diamide compounds having muscarinicreceptor antagonist and β₂ adrenergic receptor agonist activity. Theinvention also relates to pharmaceutical compositions comprising suchcompounds, processes and intermediates for preparing such compounds, andmethods of using such compounds as bronchodilating agents to treatpulmonary disorders.

2. State of the Art

Pulmonary disorders, such as chronic obstructive pulmonary disease(COPD) and asthma, are commonly treated with bronchodilators. See, forexample, Ziedalski et al., Advances in the Management of ChronicObstructive Pulmonary Disease, Expert Opin. Pharmacother., (2003) 4(7),1063-1082; Tashkin et al., The Role of Long-Acting Bronchodilators inthe Management of Stable COPD, Chest, 2004: 125; 249-259; and Donohue,Therapeutic Responses in Asthma and COPD: Bronchodilators, Chest, 2004:126; 125-137. Such bronchodilators are typically administered byinhalation using a hand-held inhaler device.

Commonly-used bronchodilating agents typically have muscarinic receptorantagonist activity (i.e., anticholinergic agents) or β₂ adrenergicreceptor (adrenoceptor) agonist activity. More recently, compoundshaving both muscarinic receptor antagonist and β₂ adrenergic receptoragonist (MABA) activities in the same molecule have been reported. Forexample, U.S. Pat. No. 7,141,671, issued Nov. 28, 2006, disclosesbiphenyl compounds having both muscarinic receptor antagonist and β₂adrenergic receptor agonist activities.

Dual-acting MABA compounds are expected to be particularly useful fortreating pulmonary disorders because such compounds can be formulatedand administered as a single therapeutic agent but, once administered,they provide bronchodilation through two distinct, and possiblysynergistic, modes of action. Additionally, MABA compounds have thepotential to be combined with an anti-inflammatory agent, such as aninhaled corticosteroid (ICS), to provide triple therapy in a singleinhaler using only two therapeutic agents (MABA+ICS).

Thus, a need exists for new MABA compounds. In particular, a need existsfor new MABA compounds that are highly effective as both a muscarinicreceptor antagonist and a β₂ adrenergic receptor agonist. Additionally,MABA compounds having a long duration of action, i.e., compounds thatprovide significant bronchodilation for at least about 24 hours afteradministration by inhalation, may be particularly useful for treatingcertain pulmonary disorders where once-daily administration of abronchodilating agent is desired.

SUMMARY OF THE INVENTION

This invention relates to novel diamide compounds having both muscarinicreceptor antagonist and β₂ adrenergic receptor agonist activities. Suchcompounds produce bronchodilation when administered to a mammal byinhalation. In some cases, compounds of this invention have been foundto possess a long duration of action, i.e., to produce bronchodilationfor at least about 24 hours after administration. Accordingly, compoundsof this invention are expected to be useful and advantageous asbronchodilating agents for treating pulmonary disorders.

In one aspect, this invention relates to a compound of formula I:

wherein

Y is a group of formula (a):

and Y is attached at the 3- or 4-position of the phenylene ring relativeto the —CH₂—(CR⁵R⁶)_(e)— group;

X is selected from —C(O)NH— and —NHC(O)—;

Ar¹ is selected from phen-1,3-ylene and phen-1,4-ylene, wherein thephenylene group is unsubstituted or substituted with 1 to 3 substituentsselected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl) and halo;

each R¹ is selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl),hydroxyl and halo;

each R² is selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl) andhalo;

each R³ is selected independently from C₁₋₃ alkyl; or two R³ groups arejoined to form C₁₋₃ alkylene, C₂₋₃ alkenylene or oxiran-2,3-diyl;

each R⁴ is selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl) andhalo;

R⁵ is selected from hydrogen, methyl and ethyl;

R⁶ is selected from hydrogen, methyl and ethyl;

R^(a) is selected from C₁₋₆ alkyl;

a is 0, 1, 2 or 3;

b is 0, 1, 2 or 3;

c is 0, 1, 2, 3 or 4;

d is 0, 1, 2 or 3;

e is 0 or 1;

n is 0 or 1;

p is 0, 1, 2, 3, 4, 5 or 6; provided that when n is 0, p is 1, 2, 3, 4,5 or 6;

q is 0, 1, 2, 3, 4, 5 or 6;

or a pharmaceutically acceptable salt thereof.

As used hereinafter, the phrase “compound of formula I” means a compoundof formula I or a pharmaceutically acceptable salt thereof; i.e., thisphrase means a compound of formula I in free base form or in apharmaceutically acceptable salt form unless otherwise indicated.

In another aspect, this invention relates to a pharmaceuticalcomposition comprising (a) a compound of formula I; (b) apharmaceutically acceptable carrier. This aspect of the inventionincludes, for example, pharmaceutical compositions suitable foradministration by inhalation.

In yet another aspect, this invention relates to a compositioncomprising (a) a compound of formula I, and (b) a steroidalanti-inflammatory agent (e.g., a corticosteroid). The term “steroidalanti-inflammatory agent” as used herein includes pharmaceuticallyacceptable salts and/or solvates of such agents unless otherwiseindicated. This invention also relates to a pharmaceutical compositioncomprising (a) a compound of formula I; (b) a steroidalanti-inflammatory agent; and (c) a pharmaceutically acceptable carrier.These aspects of the invention include, for example, compositionssuitable for administration by inhalation. In a particular embodiment,the steroidal anti-inflammatory agent is a corticosteroid (e.g., aglucocorticoid), such as fluticasone propionate or a solvate thereof orfluticasone furoate or a solvate thereof

In still another aspect, this invention relates to a method for treatinga pulmonary disorder in a patient comprising administering a compound offormula I to the patient. This aspect of the invention includes, forexample, treating chronic obstructive pulmonary disease or asthma. Alsoincluded are methods in which a steroidal anti-inflammatory agent isadministered simultaneously or sequentially with compound of formula Ito treat a pulmonary disorder.

In another aspect, this invention relates to a method for producingbronchodilation in a mammal comprising administering abronchodilation-producing amount of a compound of formula I to themammal. This aspect includes, for example, producing bronchodilation ina human.

In yet another aspect, this invention relates to a method forantagonizing a muscarinic receptor and agonizing a β₂ adrenergicreceptor in a biological system or sample comprising a muscarinicreceptor and a β₂ adrenergic receptor, the method comprising treatingthe biological system or sample with a compound of formula I. Thisaspect includes both in vivo and in vitro methods.

This invention also relates to processes and novel intermediates usefulfor preparing compounds of formula I. In one such embodiment, thisinvention relates to a compound of formula 3a:

or a salt thereof, wherein R^(x) and R^(y) are independently selectedfrom C₁₋₄ alkyl, phenyl, and —C₁₋₄ alkyl-(phenyl); and R^(z) is selectedfrom C₁₋₄ alkyl, phenyl, —C₁₋₄ alkyl-(phenyl) and —O—(C₁₋₄ alkyl).

In yet another of its method aspects, this invention relates to aprocess of preparing a compound of formula I, the process comprisingdeprotecting a compound of formula 3a to provide a compound of formulaI.

Other aspects and embodiments of this invention are described herein.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention relates to compoundsof formula I. Such compounds contain one or more chiral centers andtherefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Compounds of formula I also contain several basic groups (e.g., aminogroups) and therefore, such compounds can exist as the free base or invarious salt forms, such a mono-protonated salt form or a di-protonatedsalt form or mixtures thereof. All such forms are included within thescope of this invention, unless otherwise indicated.

This invention also includes isotopically-labeled compounds of formulaI, i.e., compounds of formula I where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula I include,but are not limited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O,³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest are compounds of formula Ienriched in tritium or carbon-14, which compounds can be used, forexample, in tissue distribution studies. Also of particular interest arecompounds of formula I enriched in deuterium especially at a site ofmetabolism, which compounds are expected to have greater metabolicstability. Also of particular interest are compounds of formula Ienriched in a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N,which compounds can be used, for example, in Positron EmissionTomography (PET) studies.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention, unlessotherwise specified.

The compounds described herein have typically been named using theAutoNom feature of the commercially-available MDL® ISIS/Draw software(Symyx, Santa Clara, Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

In one embodiment, n is 0, and Ar¹ is absent. In another embodiment, nis 1, and Ar¹ is present.

When present, in one embodiment, Ar¹ is unsubstituted phen-1,3-ylene. Inanother embodiment, Ar¹ is phen-1,3-ylene substituted with 1 to 3substituents selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl) andhalo. In another embodiment, Ar¹ is phen-1,3-ylene substituted with 1 or2 substituents selected independently from methyl, ethyl, methoxy,fluoro or chloro. Representative examples of Ar¹ include, but are notlimited to, phen-1,3-ylene, 2-methylphen-1,3-ylene,4-methylphen-1,3-ylene, 5-methylphen-1,3-ylene, 6-methylphen-1,3-ylene,2-methoxyphen-1,3-ylene, 4-methoxyphen-1,3-ylene,5-methoxyphen-1,3-ylene, 6-methoxyphen-1,3-ylene,2-fluorophen-1,3-ylene, 4-fluorophen-1,3-ylene, 5-fluorophen-1,3-ylene,6-fluorophen-1,3-ylene, 2-chlorophen-1,3-ylene, 4-chlorophen-1,3-ylene,5-chlorophen-1,3-ylene, 6-chlorophen-1,3-ylene,2,4-dimethylphen-1,3-ylene, 2,5-dimethylphen-1,3-ylene,2,6-dimethylphen-1,3-ylene, 4,6-dimethylphen-1,3-ylene,2-chloro-5-methoxyphen-1,3-ylene, and 5-chloro-2-methoxyphen-1,3-ylene.In a particular embodiment, Ar¹ is phen-1,3-ylene or6-methylphen-1,3-ylene.

In another embodiment, Ar¹ is unsubstituted phen-1,4-ylene. In yetanother embodiment, Ar¹ is phen-1,4-ylene substituted with 1 to 3substituents selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl) andhalo. In another embodiment, Ar¹ is phen-1,4-ylene substituted with 1 or2 substituents selected independently from methyl, ethyl, methoxy,fluoro or chloro. Representative examples of Ar¹ include, but are notlimited to, phen-1,4-ylene, 2-methylphen-1,4-ylene,3-methylphen-1,4-ylene, 5-methylphen-1,4-ylene, 6-methylphen-1,4-ylene,2-methoxyphen-1,4-ylene, 3-methoxyphen-1,4-ylene,5-methoxyphen-1,4-ylene, 6-methoxyphen-1,4-ylene,2-fluorophen-1,4-ylene, 3-fluorophen-1,4-ylene, 5-fluorophen-1,4-ylene,6-fluorophen-1,4-ylene, 2-chlorophen-1,4-ylene, 3-chlorophen-1,4-ylene,5-chlorophen-1,4-ylene, 6-chlorophen-1,4-ylene,2,3-dimethylphen-1,4-ylene, 2,5-dimethylphen-1,4-ylene,2,6-dimethylphen-1,4-ylene, 3,5-dimethylphen-1,4-ylene,2-chloro-5-methoxyphen-1,4-ylene, and 5-chloro-2-methoxyphen-1,4-ylene.

In a particular embodiment, Ar¹ is phen-1,3-ylene, phen-1,4-ylene or6-methylphen-1,3-ylene.

In separate embodiments, p is 0, 1, 2, 3, 4, 5 or 6; provided that pcannot be 0 when n is 0. Additionally, in one embodiment, when n is 0and X is —NHC(O)—, then p is typically not 1. Representative —(CH₂)_(p)—groups include —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, and—(CH₂)₆—. In a particular embodiment, p is 0, 1, 2, 3, or 4.

In separate embodiments, q is 0, 1, 2, 3, 4, 5 or 6. Representative—(CH₂)_(q)— groups include —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—,—(CH₂)₅—, and —(CH₂)₆—. In a particular embodiment, q is 0, 1 or 2.

In one embodiment, X is —C(O)NH—. In another embodiment, X is —NHC(O)—.

In a particular embodiment, Y forms a group of the formula:—(CH₂)_(p)—X—(CH₂)_(q)—, where X, p and q are as defined herein.Representative examples include:

—(CH₂)_(p)—C(O)NH—(CH₂)_(q)—; and

—(CH₂)_(p)—NHC(O)—(CH₂)_(q)—.

In another particular embodiment, Y forms a group of the formula:—(CH₂)_(p)—X—, where X and p are as defined herein. Representativeexamples include:

—(CH₂)_(p)—C(O)NH—; and

—(CH₂)_(p)—NHC(O)—.

In another particular embodiment, Y forms a group of the formula:—Ar¹—(CH₂)_(p)—X—(CH₂)_(q)—, where Ar¹, X, p and q are as definedherein. Representative examples include:

-(phen-1,3-ylene)-(CH₂)_(p)—C(O)NH—(CH₂)_(q)—;

-(phen-1,3-ylene)-(CH₂)_(p)—NHC(O)—(CH₂)_(q)—;

-(phen-1,4-ylene)-(CH₂)_(p)—C(O)NH—(CH₂)_(q)—; and

-(phen-1,4-ylene)-(CH₂)_(p)—NHC(O)—(CH₂)_(q)—;

where the phen-1,3-ylene or phen-1,4-ylene group is unsubstituted orsubstituted as defined herein.

In another particular embodiment, Y forms a group of the formula:—Ar¹—(CH₂)_(p)—X—, where Ar¹, X and p are as defined herein.Representative examples include:

-(phen-1,3-ylene)-(CH₂)_(p)—C(O)NH—;

-(phen-1,3-ylene)-(CH₂)_(p)—NHC(O)—;

-(phen-1,4-ylene)-(CH₂)_(p)—C(O)NH—; and

-(phen-1,4-ylene)-(CH₂)_(p)—NHC(O)—;

where the phen-1,3-ylene or phen-1,4-ylene group is unsubstituted orsubstituted as defined herein.

In another particular embodiment, Y forms a group of the formula:—Ar¹—X—(CH₂)_(q)—, where Ar¹, X and q are as defined herein.Representative examples include:

-(phen-1,3-ylene)-C(O)NH—(CH₂)_(q)—;

-(phen-1,3-ylene)-NHC(O)—(CH₂)_(q)—;

-(phen-1,4-ylene)-C(O)NH—(CH₂)_(q)—; and

-(phen-1,4-ylene)-NHC(O)—(CH₂)_(q)—;

where the phen-1,3-ylene or phen-1,4-ylene group is unsubstituted orsubstituted as defined herein.

In another particular embodiment, Y forms a group of the formula:—Ar¹—X—, where Ar¹ and X are as defined herein. Representative examplesinclude:

-(phen-1,3-ylene)-C(O)NH—;

-(phen-1,3-ylene)-NHC(O)—;

-(phen-1,4-ylene)-C(O)NH—; and

-(phen-1,4-ylene)-NHC(O)—;

where the phen-1,3-ylene or phen-1,4-ylene group is unsubstituted orsubstituted as defined herein.

In one embodiment, Y is attached at the 3-position of the phenylene ringrelative to the —CH₂—(CR⁵R⁶)_(e)— group. In another embodiment, Y isattached at the 4-position of the phenylene ring relative to the—CH₂—(CR⁵R⁶)_(e)— group.

In one embodiment, a is 0, and R¹ is absent. In other separateembodiments, a is 1, 2 or 3, i.e., one, two or three R¹ groups arepresent at any available position of the phenyl ring to which R¹ isattached. For example, when a is 1, R¹ can be at the 2-, 3-, 4-, 5- or6-position of the phenyl ring to which R¹ is attached; when a is 2, R¹groups can be at the 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, or 3,5-positions ofthe phenyl ring to which R¹ is attached; and when a is 3, R¹ groups canbe at the 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6-, or 3,4,5-positions ofthe phenyl ring to which R¹ is attached.

When present, in one embodiment, each R¹ is selected independently fromC₁₋₃ alkyl, —O—(C₁₋₃ alkyl), hydroxyl and halo. In another embodiment,each R¹ is selected independently from C₁₋₃ alkyl, —O—(C₁₋₃ alkyl), andhalo. In another embodiment, each R¹ is selected independently frommethyl, ethyl, methoxy, fluoro, chloro and bromo.

In one embodiment, b is 0, and R² is absent. In other separateembodiments, b is 1, 2 or 3, i.e., one, two or three R² groups arepresent at any available position of the phenylene ring to which R² isattached. For example, when b is 1, an R² group can be at the 3-, 4-, 5-or 6-position of the phen-1,2-ylene ring to which R² is attached; when bis 2, R² groups can be at the 3,4-, 3,5-, 3,6-, 4,5-, 4,6-, or5,6-positions of the phen-1,2-ylene ring to which R² is attached; andwhen b is 3, R² groups can be at the 3,4,5-, 3,4,6-, or 4,5,6-positionsof the phen-1,2-ylene ring to which R² is attached.

When present, in one embodiment, each R² is selected independently fromC₁₋₃ alkyl, —O—(C₁₋₃ alkyl), and halo. In another embodiment, each R² isselected independently from halo. In another embodiment, each R² isselected independently from methyl, ethyl, methoxy, fluoro, chloro andbromo.

In one embodiment, c is 0, and R³ is absent. In other separateembodiments, c is 1, 2, 3 or 4, i.e., one, two, three or four R³ groupsare present at any available position of the piperidin-1,4-yl ring towhich R³ is attached.

When present, in one embodiment, each R³ is selected independently fromC₁₋₃ alkyl. In another embodiment, each R³ is methyl. Representative R³groups include methyl, ethyl, n-propyl and isopropyl.

In another embodiment, two R³ groups are joined to form C₁₋₃ alkylene orC₂₋₃ alkenylene or oxiran-2,3-diyl. Representative groups include —CH₂—,—CH₂CH₂—, —CH═CH—, —CH₂CH₂CH₂—, and —CH₂CH═CH—. For example, two R³groups at the 2- and 6-positions on the piperidine ring can be joined toform an ethylene bridge (i.e., the piperidine ring and the R³ groupsform an 8-azabicyclo[3.2.1]octane ring); or two R³ groups at the 1- and4-positions on the piperidine ring can be joined to form an ethylenebridge (i.e., the piperidine ring and the R³ groups form an1-azabicyclo[2.2.2]octane ring); or two R³ groups at the 2- and6-positions on the piperidine ring can be joined to form an ethenylenebridge (i.e., the piperidine ring and the R³ groups form an8-azabicyclo[3.2.1]oct-6-ene ring). In this embodiment, other R³ groupsas defined herein may also be present.

In still another embodiment, two R³ groups are joined to form anoxiran-2,3-diyl group. For example, two R³ groups at the 2- and6-positions on the piperidine ring can be joined to form a3-oxatricyclo[3.3.1.0^(2,4)]nonane ring. In this embodiment, other R³groups as defined herein may also be present.

In one embodiment, d is 0, and R⁴ is absent.

In other separate embodiments, d is 1, 2, or 3, i.e., one, two or threeR⁴ groups may be attached at any available position of the phenylenering to which R⁴ is attached. For example, when d is 1 and Y is attachedat the 3-position of the phenylene ring, an R⁴ group can be at the 2-,4-, 5- or 6-position of the phen-1,3-ylene ring to which R⁴ is attached.When d is 1 and Y is attached at the 4-position of the phenylene ring,an R⁴ group can be at, for example, the 2-, 3-, 5- or 6-position of thephen-1,4-ylene ring to which R⁴ is attached.

When d is 2 and Y is attached at the 3-position of the phenylene ring,R⁴ groups can be at, for example, the 2,4-, 2,5-, 2,6-, 4,5-, 4,6-, or5,6-positions of the phen-1,3-ylene ring to which R⁴ is attached. When dis 2 and Y is attached at the 4-position of the phenylene ring, R⁴groups can be at, for example, the 2,3-, 2,5-, 2,6-, 3,5-, 3,6-, or5,6-positions of the phen-1,4-ylene ring to which R⁴ is attached.

When d is 3 and Y is attached at the 3-position of the phenylene ring,R⁴ groups can be at, for example, the 2,4,5-, 2,4,6-, 2,5,6-, or4,5,6-positions of the phenylene ring to which R⁴ is attached. When d is3 and Y is attached at the 4-position of the phenylene ring, R⁴ groupscan be at, for example, the 2,3,5-, 2,3,6-, 2,5,6-, or 3,5,6-positionsof the phenylene ring to which R⁴ is attached.

When present, in one embodiment, each R⁴ is selected independently fromC₁₋₃ alkyl, —O—(C₁₋₃ alkyl), and halo. In another embodiment, each R⁴ isselected independently from methyl, ethyl, methoxy, fluoro, chloro andbromo. In a particular embodiment, R⁴ is selected from methyl, methoxy,chloro and fluoro. In another particular embodiment, d is 2, and each R⁴is methyl. In yet another particular embodiment, d is 2, and one R⁴ ismethoxy, and the other R⁴ is chloro.

In one embodiment, e is 0, and —CR⁵R⁶— is absent. In another embodiment,e is 1.

When present, in one embodiment, R⁵ is hydrogen. In another embodiment,R⁵ is methyl. In still another embodiment, R⁵ is ethyl.

When present, in one embodiment, R⁶ is hydrogen. In another embodiment,R⁶ is methyl. In still another embodiment, R⁶ is ethyl. In a particularembodiment, R⁶ is hydrogen, and R⁵ is hydrogen or methyl. In anotherparticular embodiment, R⁵ and R⁶ are both methyl.

When R⁵≠R⁶, the carbon to which R⁵ and R⁶ are attached is chiral. In oneembodiment, this stereocenter has the (R)-configuration. In anotherembodiment, this stereocenter has the (S)-configuration. In particularembodiments, the group —CH₂—(CR⁵R⁶)_(e)— is selected from —CH₂—,—CH₂CH₂—, —CH₂C*H(CH₃)—, where C* has the (R) configuration, the (S)configuration or is racemic.

In one embodiment, R^(a) is selected from C₁₋₆ alkyl. In anotherembodiment, R^(a) is C₁₋₄ alkyl. In still another embodiment, R^(a) isC₁₋₃ alkyl. Representative R^(a) groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl andn-hexyl. In a particular embodiment, R^(a) is methyl.

In one embodiment, the compound of formula I is a free base. In anotherembodiment, the compound of formula I is a mono-salt form. In yetanother embodiment, the compound of formula I is a di-salt form.

Representative Subgeneric Groupings

The following subgeneric formulae and groupings are intended to providerepresentative examples of various aspects and embodiments of thisinvention and as such, they are not intended to exclude otherembodiments or to limit the scope of the embodiments or the invention,unless otherwise indicated.

A particular embodiment of this invention relates to a compound offormula Ia:

i.e., a compound of formula I wherein a, b and c are 0 (i.e., R¹, R² andR³ are absent), R⁶ is hydrogen, and R⁴, R⁵, R^(a), Y, d and e are asdefined herein, or a pharmaceutically acceptable salt thereof.

Another particular embodiment relates to a compound of formula Ib:

i.e., a compound of formula I wherein a, b, c and e are 0 (i.e., R¹, R²,R³ and CR⁵R⁶ are absent), and R⁴, R^(a), Y and d are as defined herein,or a pharmaceutically acceptable salt thereof.

Another particular embodiment relates to a compound of formula Ic:

i.e., a compound of formula I wherein a, b and c are 0 (i.e., R¹, R² andR³ are absent), R⁶ is hydrogen, e is 1, and R⁴, R⁵, R^(a), Y and d areas defined herein, or a pharmaceutically acceptable salt thereof.

Another particular embodiment relates to a compound of formula II:

i.e., a compound of formula I wherein a, b, c and e are 0 (i.e., R¹, R²,R³ and CR⁵R⁶ are absent), Y forms a group of the formula:—(CH₂)_(p)—C(O)NH—, and R⁴, R^(a), d and p are as defined herein, or apharmaceutically acceptable salt thereof.

In a particular embodiment of compounds of formula II, R^(a) is methyl;p is 3 or 4; and d is 0, 1 or 2; and each R⁴ is selected independentlyfrom methyl; methoxy, chloro and fluoro.

In another particular embodiment of compounds of formula II, R^(a) ismethyl; p is 4; and d is 0, 1 or 2; and each R⁴ is selectedindependently from methyl; methoxy, chloro and fluoro. As shown in TableIII, all compounds of this embodiment tested in the rat Einthoven assay(100 μg) exhibited a bronchoprotective effect at 24 h.

Another particular embodiment relates to a compound of formula III:

i.e., a compound of formula I wherein a, b and c are 0 (i.e., R¹, R² andR³ are absent), R⁶ is hydrogen, e is 1, Y forms a group of the formula:—(CH₂)_(p)—NHC(O)—(CH₂)_(q)— and R⁴, R⁵, R^(a), d, p and q are asdefined herein, or a pharmaceutically acceptable salt thereof.

In a particular embodiment of compounds of formula III, R^(a) is methyl;p is 3 or 4; q is 1; R⁵ is methyl; and d is 0.

In another embodiment, this invention relates to a compound of formulaIV:

i.e., a compound of formula I wherein a, b and c are 0 (i.e., R¹, R² andR³ are absent), R⁶ is hydrogen, e is 1, Y forms a group of the formula:—Ar¹—(CH₂)_(p)—C(O)NH—(CH₂)_(q)—, Ar¹ is phenyl-1,4-ene, and R⁴, R⁵,R^(a), d, p and q are as defined herein, or a pharmaceuticallyacceptable salt thereof.

In a particular embodiment of compounds of formula IV, R^(a) is methyl;p is 1; q is 1 or 2; R⁵ is hydrogen or methyl; d is 0 or 1; and R⁴ isselected from methyl; methoxy, and fluoro.

In another embodiment, this invention relates to a compound of formulaV:

i.e., a compound of formula I wherein a, b and c are 0 (i.e., R¹, R² andR³ are absent), R⁶ is hydrogen, e is 1, Y forms a group of the formula:—Ar¹—C(O)NH—, Ar¹ is phenyl-1,3-ene, and R⁴, R⁵, R^(a), and d are asdefined herein, or a pharmaceutically acceptable salt thereof

In a particular embodiment of compounds of formula V, R^(a) is methyl;R⁵ is hydrogen or methyl; d is 0, 1 or 2; and each R⁴ is selectedindependently from methyl; methoxy, chloro and fluoro.

Particular embodiments of compounds of formula I are compounds where a,b and c are 0 (i.e., R¹, R² and R³ are absent), R^(a) is methyl; and(Ar¹)_(n), (CH₂)_(p), X, (CH₂)_(q), (R⁴)_(d) (and the phenylene ring towhich it is attached), and —CH₂(CR⁵R⁶)_(e)— are as defined in Table I:

TABLE I ID (Ar¹)_(n) (CH₂)_(p) X (CH₂)_(q)

I-1  — (CH₂)₃ C(O)NH —

CH₂ I-2  — (CH₂)₃ C(O)NH —

CH₂ I-3  — (CH₂)₃ C(O)NH —

CH₂ I-4  — (CH₂)₃ C(O)NH —

CH₂ I-5  — (CH₂)₃ C(O)NH —

CH₂ I-6  — (CH₂)₃ C(O)NH —

CH₂ I-7  — (CH₂)₃ C(O)NH —

(CH₂)₂ I-8  — (CH₂)₃ C(O)NH —

(CH₂)₂ I-9  — (CH₂)₃ C(O)NH —

(CH₂)₂ I-10 — (CH₂)₃ C(O)NH —

I-11 — (CH₂)₃ NHC(O) CH₂

I-12 — (CH₂)₃ NHC(O) CH₂

I-13 — (CH₂)₃ NHC(O) CH₂

I-14 — (CH₂)₄ C(O)NH —

CH₂ I-15 — (CH₂)₄ C(O)NH —

CH₂ I-16 — (CH₂)₄ C(O)NH —

CH₂ I-17 — (CH₂)₄ C(O)NH —

CH₂ I-18 — (CH₂)₄ C(O)NH —

CH₂ I-19 — (CH₂)₄ C(O)NH —

CH₂ I-20 — (CH₂)₄ C(O)NH —

(CH₂)₂ I-21 — (CH₂)₄ C(O)NH —

(CH₂)₂ I-22 — (CH₂)₄ NHC(O) CH₂

I-23 — (CH₂)₄ NHC(O) CH₂

I-24 — (CH₂)₄ NHC(O) CH₂

I-25

— C(O)NH —

CH₂ I-26

— C(O)NH —

CH₂ I-27

— C(O)NH —

CH₂ I-28

— C(O)NH —

CH₂ I-29

— C(O)NH —

CH₂ I-30

— C(O)NH —

CH₂ I-31

— C(O)NH —

CH₂ I-32

— C(O)NH —

(CH₂)₂ I-33

— C(O)NH —

(CH₂)₂ I-34

— C(O)NH —

(CH₂)₂ I-35

— C(O)NH —

(CH₂)₂ I-36

— C(O)NH —

I-37

— C(O)NH —

I-38

— C(O)NH —

I-39

— C(O)NH —

I-40

— C(O)NH —

I-41

— C(O)NH —

I-42

— C(O)NH —

I-43

— C(O)NH —

I-44

— C(O)NH CH₂

I-45

— C(O)NH CH₂

I-46

— C(O)NH CH₂

I-47

— C(O)NH CH₂

I-48

— C(O)NH CH₂

I-49

— C(O)NH CH₂

I-50

— C(O)NH CH₂

I-51

— C(O)NH CH₂

I-52

— NHC(O) CH₂

I-53

— NHC(O) CH₂

I-54

— NHC(O) CH₂

I-55

— C(O)NH (CH₂)₂

I-56

— C(O)NH (CH₂)₂

I-57

— C(O)NH (CH₂)₂

I-58

— C(O)NH (CH₂)₂

I-59

CH₂ C(O)NH —

CH₂ I-60

CH₂ C(O)NH —

CH₂ I-61

CH₂ C(O)NH —

CH₂ I-62

CH₂ C(O)NH —

(CH₂)₂ I-63

CH₂ C(O)NH —

(CH₂)₂ I-64

CH₂ C(O)NH CH₂

(CH₂)₂ I-65

CH₂ C(O)NH CH₂

(CH₂)₂ I-66

CH₂ C(O)NH CH₂

I-67

CH₂ C(O)NH CH₂

I-68

CH₂ C(O)NH CH₂

I-69

CH₂ C(O)NH CH₂

I-70

CH₂ C(O)NH CH₂

I-71

CH₂ C(O)NH CH₂

I-72

CH₂ C(O)NH CH₂

I-73

CH₂ C(O)NH CH₂

I-74

CH₂ NHC(O) CH₂

CH₂ I-75

CH₂ NHC(O) CH₂

CH₂ I-76

CH₂ NHC(O) CH₂

(CH₂)₂ I-77

CH₂ NHC(O) CH₂

I-78

CH₂ NHC(O) CH₂

I-79

CH₂ NHC(O) CH₂

I-80

CH₂ NHC(O) CH₂

I-81

CH₂ NHC(O) CH₂

I-82

CH₂ NHC(O) CH₂

I-83

CH₂ NHC(O) CH₂

I-84

CH₂ NHC(O) CH₂

I-85

CH₂ C(O)NH (CH₂)₂

I-86

CH₂ C(O)NH (CH₂)₂

I-87

CH₂ C(O)NH (CH₂)₂

I-88

CH₂ C(O)NH (CH₂)₂

I-89

CH₂ C(O)NH (CH₂)₂

I-90

CH₂ C(O)NH (CH₂)₂

I-91

CH₂ NHC(O) (CH₂)₂

I-92

CH₂ NHC(O) (CH₂)₂

I-93

CH₂ NHC(O) (CH₂)₂

I-94

CH₂ NHC(O) (CH₂)₂

I-95

CH₂ NHC(O) (CH₂)₂

I-96

(CH₂)₂ NHC(O) CH₂

CH₂

The compounds listed in Table I may be in free base form or may be in apharmaceutically acceptable salt form. Of particular interest arecompounds of Table I that demonstrate a bronchoprotective effect 24hours after administration by inhalation, e.g., as determined in the ratEinthoven Assay.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings unlessotherwise indicated.

The singular terms “a,” “an” and “the” include the corresponding pluralterms unless the context of use clearly dictates otherwise.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), benzyl, formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxyl-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxyl group (i.e., —COOH).Representative carboxyl-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, tert-butyl, benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS, TBDMS), diphenylmethyl (benzhydryl, DPM)and the like.

The term “halo” means fluoro, chloro, bromo, and iodo.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(C₁₋₆ alkyl)silyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including C₁₋₆ alkanoyl groups, such as formyl, acetyl andthe like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and the like.Additionally, two hydroxyl groups can also be protected as an alkylidenegroup, such as prop-2-ylidine, formed, for example, by reaction with aketone, such as acetone.

The term “leaving group” means a functional group or an atom that can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include, but are not limited to,chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate,tosylate, brosylate, nosylate and the like; and acyloxy groups, such asacetoxy, trifluoroacetoxy and the like.

The term “micronized” or “in micronized form” means particles in whichat least about 90 percent of the particles have a diameter of less thanabout 10 μm unless otherwise indicated.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected or blocked from undergoing undesired reactionswith a protecting or blocking group. Functional groups that may beprotected include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like. Suitableprotecting groups for such functional groups are well known to those ofordinary skill in the art as exemplified by the teachings in T. W.Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and the references cited therein.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula I, i.e. where one or more amino groups having beenprotonated by an acid. Typically, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD or asthma) ina patient, such as a mammal (e.g., a human), that includes any of thefollowing or combinations thereof:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

Compounds of this invention, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., R¹, R², Y, a, b, etc.)used in the following schemes have the same meanings as those definedelsewhere herein unless otherwise indicated. Additionally, compoundshaving an acidic or basic atom or functional group may be used or may beproduced as a salt unless otherwise indicated (in some cases, the use ofa salt in a particular reaction will require conversion of the salt to anon-salt form, e.g., a free base, using routine procedures beforeconducting the reaction).

Scheme 1 illustrates a typical procedure for preparing compounds offormula (where R⁶ is hydrogen):

wherein

G¹ is —CHO or —CH₂C(O)R⁵; and

P¹ is a hydroxyl-protecting group, such as tert-butyldimethylsilyl.

In this procedure, compound 1 is reacted with about 0.95 to about 1.5molar equivalents of compound 2 in the presence of a reducing agent toafford compound 3. Any suitable reducing agent may be used in thisreaction including, by way of illustration, a metal hydride reagent,such as sodium borohydride, sodium triacetoxyborohydride, sodiumcyanoborohydride and the like, or hydrogen and a metal catalyst, such aspalladium on carbon, and the like. This reaction is typically conductedat a temperature ranging from about −20° C. to about 30° C. (e.g., about0° C. to about 5° C.) for about 1 hour to about 6 hours or until thereaction is substantially complete. Typically, this reaction isconducted in a diluent, such as dichloromethane (DCM), dichloroethaneand the like. Optionally, the diluent may contain a protic solvent, suchas methanol and the like. Upon completion of the reaction, the productis typically isolated using conventional procedures, such as extraction,recrystallization, chromatography and the like. Alternatively, ifdesired, the reaction mixture containing compound 3 can be used directlyin the next step of the synthesis without further isolation orpurification.

Compound 3 is then deprotected to provide a compound of formula 1. Theparticular conditions used to deprotect compound 3 will depend on theprotecting group employed. For example, when P¹ is a silyl protectinggroup, such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl,diphenylmethylsilyl, di-tert-butylmethylsilyl, tert-butoxydiphenylsilyl,and the like (i.e., a compound of formula 3a as defined herein), thisdeprotection reaction is typically conducted by contacting compound 3with a source of fluoride ion. In a particular embodiment, the source offluoride ion is triethylamine trihydrofluoride. Other suitable sourcesof fluoride ion include tetrabutylammonium fluoride, potassium fluoridewith 18-crown-6, hydrogen fluoride, pyridine hydrofluoride, and thelike. This reaction is typically conducted at a temperature ranging fromabout 0° C. to about 50° C., (e.g., about 10° C. to about 25° C.) forabout 24 to about 72 hours or until the reaction is substantiallycomplete. Typically, this reaction is conducted in a diluent, such asDCM, dichloroethane and the like. Upon completion of the reaction, theproduct is typically isolated using conventional procedures, such asextraction, recrystallization, chromatography and the like.

Compounds of formula 1 are typically prepared by deprotecting thecorresponding acetal or ketal intermediate. For example, when G¹ is—CHO, compounds of formula 1 are typically prepared by deprotecting anintermediate of formula 4a:

wherein P^(2a) and P^(2b) are selected independently from C₁₋₆ alkyl, orP^(2a) and P^(2b) are joined to form C₂₋₆ alkylene, typically C₂₋₄alkylene.

Similarly, when G¹ is —CH₂C(O)R⁵, compounds of formula 1 are typicallyprepared by deprotecting an intermediate of formula 4b:

wherein P^(3a) and P^(3b) are selected independently from C₁₋₆ alkyl, orP^(3a) and P^(3b) are joined to form C₂₋₆ alkylene, typically C₂₋₄alkylene.

Deprotection of compound 4a or 4b is typically conducted by reacting 4aor 4b with aqueous acid to hydrolyze the acetal or ketal group andprovide the corresponding aldehyde or ketone compound 1. Any suitableacid can be employed in this reaction including, by way of example,hydrochloric acid, sulfuric acid, methanesulfonic acid,p-toluenesulfonic acid and the like. The hydrolysis reaction istypically conducted at a temperature ranging from about 0° C. to about30° C. (e.g., about 20° C. to about 25° C.) for about 1 to about 6 hoursor until the reaction is substantially complete. Typically, thisreaction is conducted in a diluent, such as methanol, ethanol,isopropanol, dichloromethane/ethanol, acetonitrile and the like. Uponcompletion of the reaction, the product is typically isolated usingconventional procedures, such as extraction, recrystallization,chromatography and the like. Alternatively, the reaction mixturecontaining compound 1 can be used directly in the next step of thesynthesis.

Compounds of formula 4a or 4b are typically prepared by coupling acompound of formula 5:

with a compound of formula 6a or 6b:

wherein

G² is —NH₂ and G³ is —COOH; or G² is —COOH and G³ is —NH₂.

The coupling reaction between compound 5 and compound 6a or 6b to formcompound 4a or 4b is typically conducted using a carboxylic acid—aminecoupling reagent. Any suitable carboxylic acid—amine coupling reagentmay be used in this reaction including, by way of illustration,benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate (BOP); N,N-carbonyldiimidazole (CDI);dicyclohexylcarbodiimide (DCC);3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT);1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC HCl);2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU);2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU); 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU); 1-hydroxy-7-azabenzotriazole (HOAt);N-hydroxybenzotriazole (HOBt);benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate(PyBOP); bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrOP);O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU);2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU);N,N,N′,N′-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)uraniumtetrafluoroborate (TDBTU); O—(N-succinimidyl)-1,1,3,3-tetramethyluranium tetrafluoroborate (TSTU); and combinations thereof, such as EDCand HOBt.

The coupling reaction is typically conducted by reacting about 0.95 toabout 1.5 molar equivalents of the amine compound (5, when G² is —NH₂;or 6a or 6b when G³ is —NH₂) with the carboxylic acid (5, when G² is—COOH; or 6a or 6b when G³ is —COOH) in the presence of the couplingreagent. The coupling reagent is typically used in an amount rangingfrom about 1.0 to about 1.5 molar equivalents relative to the carboxylicacid. Generally, this reaction is conducted in the presence of ahindered amine, such as diisopropylethylamine (DIEA), N-methylmorpholine(NMM), collidine, 2,3,5,6-tetramethylpyridine (TEMP),2,6-di-tert-butyl-4-dimethylaminopyridine (DBDMAP) and the like, in adiluent, such as dichloromethane, tetrahydrofuran, 1,4-dioxane,acetonitrile, dimethylformamide, dimethyl acetamide, N-methylpyrrolidoneor mixtures thereof. The reaction is typically conducted at atemperature ranging from about −20° C. to about 50° C. (e.g., about 20°C. to about 25° C.) for about 1 to about 30 hours or until the reactionis substantially complete. Upon completion of the reaction, the productis typically isolated using conventional procedures, such as extraction,recrystallization, chromatography, and the like.

Compounds of formula 5 are typically prepared by coupling a compound offormula 7:

with a compound of formula 8a, 8b or 8c:

wherein P⁴ is a carboxyl-protecting group (such as C₁₋₆ alkyl, includingmethyl, ethyl, n-propyl and the like; or benzyl); and P⁵ is anamino-protecting group (such as BOC, Fmoc, Cbz and the like). When acompound of formula 8c is used, the nitro group is subsequently reducedto an amino group using standard reagents and procedures, such as zinc,tin or iron metal and acid (such as acetic acid, hydrochloric acid andthe like), or catalytic hydrogenation. In this embodiment, p istypically 0.

Compounds of formula 8a, 8b and 8c are commercially available, known inthe art or can be prepared using routine variations of procedures knownin the art.

Representative compounds of formula 8a include, by way of example,methyl 4-(methylamino)butyrate, methyl 5-(methylamino)pentanoate, methyl3-(methylamino)benzoate, methyl 4-(methylamino)benzoate, methyl3-(methylamino)-4-methylbenzoate, methyl [3-(methylamino)phenyl]acetateand the like.

Representative compounds of formula 8b include, by way of example,(3-methylaminopropyl)carbamic acid tert-butyl ester,(3-methylaminopropyl)carbamic acid 9H-fluoren-9-ylmethyl ester,(4-methylaminobutyl)carbamic acid tert-butyl ester,(4-methylaminobutyl)carbamic acid 9H-fluoren-9-ylmethyl ester,(3-methylaminophenyl)carbamic acid tert-butyl ester,(3-methylaminophenyl)carbamic acid 9H-fluoren-9-ylmethyl ester,(4-methylaminophenyl)carbamic acid tert-butyl ester,(4-methylaminophenyl)carbamic acid 9H-fluoren-9-ylmethyl ester,(3-methylaminobenzyl)carbamic acid tert-butyl ester,(3-methylaminobenzyl)carbamic acid 9H-fluoren-9-ylmethyl ester,(4-methylaminobenzyl)carbamic acid tert-butyl ester,(4-methylaminobenzyl)carbamic acid 9H-fluoren-9-ylmethyl ester, and thelike.

Representative compounds of formula 8c include, by way of example,N-methyl-3-nitroaniline, N-methyl-4-nitroaniline,N-ethyl-3-nitroaniline, N-ethyl-4-nitroaniline, and the like.

The carboxylic acid—amine coupling reaction between compound 7 andcompound 8a or 8b to form compound 5 is typically conducted using thereagents and reaction conditions described herein for coupling acarboxylic acid and an amine (e.g., compound 5 and compound 6a or 6b).Upon completion of the reaction, the product is typically isolated usingconventional procedures, such as extraction, recrystallization,chromatography, and the like.

Compounds of formula 7 are typically prepared by reacting a compound offormula 9:

with about 0.95 to about 1.5 molar equivalents of acrylic acid. Thisreaction is typically conducted in a diluent, such as dichloromethane,at a temperature ranging from about 20° C. to about 70° C. (e.g., about50° C.) for about 6 to about 30 hours or until the reaction issubstantially complete. Upon completion of the reaction, the product istypically isolated using conventional procedures, such as extraction,recrystallization, chromatography, and the like.

Compounds of formula 9 are known in the art or can be prepared usingroutine variations of procedures known in the art. For example,procedures for preparing such compounds are found in U.S. PatentApplication Publication No. U.S. 2004/0167167 A1 and R. Naito et al.,Chem. Pharm. Bull., 46(8) 1286-1294 (1998). By way of illustration,compounds of formula 9 are typically prepared by reacting a compound offormula 10:

with an alcohol of formula 11:

wherein P⁶ is an amino-protecting group, such as benzyl, BOC, Fmoc, Cbzand the like.

Representative compounds of formula 10 include, by way of example,2-(phenyl)phenyl isocyanate, 2-(phenyl)-5-methylphenyl isocyanate,2-(3-chlorophenyl)-4,6-difluorophenyl isocyanate,2-(phenyl)-6-fluorophenyl isocyanate, 2-(phenyl)-5-bromophenylisocyanate, 2-(4-bromophenyl)-5-bromophenyl isocyanate,2-(phenyl)-4-methoxyphenyl isocyanate, 2-(4-methoxyphenyl)phenylisocyanate, 2-(phenyl)-5-methoxyphenyl isocyanate, and the like.

Representative compounds of formula 11 include, by way of example,4-hydroxy-1-benzylpiperidine, 4-hydroxypiperidine-1-carboxylic acidtert-butyl ester, 4-hydroxypiperidine-1-carboxylic acid9H-fluoren-9-ylmethyl ester, 4-hydroxy-4-methyl-1-benzylpiperidine,2-benzyl-2-azabicyclo[2.2.1]heptan-5-ol,2-benzyl-2-azabicyclo[2.2.2]octan-5-ol,8-benzyl-8-azabicyclo[3.2.1]oct-6-en-3-ol,3-benzyl-3-azabicyclo[3.2.1]octan-8-ol,8-benzyl-8-azabicyclo[3.2.1]octan-3-ol,3-benzyl-3-azabicyclo[3.3.1]nonan-9-ol,9-benzyl-9-azabicyclo[3.3.1]nonan-3-ol, 8-benzylnortropine,8-benzylnorpseudotropine, and the like.

This reaction is typically conducted by reacting 10 with about 0.95 toabout 1.2 molar equivalents of 11 at a temperature ranging from about20° C. to about 100° C. (e.g., about 60° C. to about 80° C.) for about 6to about 24 hours or until the reaction is substantially complete. Ifdesired, this reaction can be conducted in a diluent, such asdichloromethane, toluene and the like. Alternatively, this reaction canbe conducted in the absence of a diluent. Upon completion of thereaction, the product is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography andthe like. Alternatively, the reaction mixture is used directly in thenext step of the synthesis without isolation of the product.

The amino-protecting group, P⁶, is then removed using conventionalprocedures to afford compound 9. For example, when P⁶ is a benzyl group,the deprotection reaction is typically conducted using hydrogen orammonium formate, in the presence of a catalyst, such as a palladiumcatalyst. Representative catalysts include, by way of illustration,palladium on carbon, palladium hydroxide on carbon and the like. Thisreaction is typically conducted at a temperature ranging from about 20°C. to about 50° C. (e.g., about 40° C.) for about 6 to about 24 hours oruntil the reaction is substantially complete. Typically, this reactionis conducted in a diluent, such as methanol, ethanol, isopropanol andthe like. Upon completion of the reaction, compound 9 is typicallyisolated using conventional procedures, such as extraction,recrystallization, chromatography and the like.

Compounds of formula 2 are known in the art or can be prepared fromcommercially available starting materials and reagents using knownprocedures. For example, the preparation of a compound of formula 2,where P¹ is tert-butyldimethylsilyl, is described in U.S. PatentApplication Publication No. 2006/0035931, published Feb. 16, 2006. Otherprotecting groups that can be employed for P¹ include, for example,dimethylisopropylsilyl, diethylisopropylsilyl, dimethylhexylsilyl,tert-butyldiphenylsilyl, diphenylmethylsilyl and the like.

Additionally, if desired, the hydroxyl group of 2 can also be protected,i.e., a compound of formula 2a can be used:

where P^(1a) is a hydroxyl protecting group, such as benzyl group or4-methoxybenzyl. For example, the preparation of a compound of formula2a, where P¹ is tert-butyldimethylsilyl and the hydroxyl group isprotected as a 4-methoxybenzyl group is described in WO 2008/096129.When 2a is used, intermediates such as 3, 12 and the like, willtypically contain the P^(1a) group. The P^(1a) is subsequently removedusing conventional procedures and reagents. For example, when P^(1a) isa benzyl group, the deprotection reaction is typically conducted usinghydrogen or ammonium formate, in the presence of a catalyst, such as apalladium catalyst. When P^(1a) is 4-methoxybenzyl, this group can beremoved under acidic hydrolysis conditions, such as 30% TFA in DCM.

Alternatively, compounds of formula I are prepared by coupling acompound of formula 5 with a compound of formula 12:

wherein G⁵ is —COOH (when G² is —NH₂); or G⁵ is —NH₂ (when G² is —COOH).

The carboxylic acid—amine coupling reaction between compound 5 andcompound 12 is typically conducted using the reagents and reactionconditions described herein for coupling a carboxylic acid and an amine(e.g., compound 5 and compound 6a or 6b). Upon completion of thereaction, the product is typically isolated using conventionalprocedures, such as extraction, recrystallization, chromatography, andthe like. Deprotection of the resulting product (e.g., removal of P¹)using standard reagents and conditions then provides a compound offormula I.

Compounds of formula 12 are prepared by reacting a compound of formula 2with a compound of formula 13a or 13b:

in the presence of a reducing agent; wherein G^(5P) is selected from:

—COOP⁷, where P⁷ is a carboxyl-protecting group (such as C₁₋₆ alkyl,including methyl, ethyl, n-propyl and the like; or benzyl); and

—NHP⁸, where P⁸ is an amino-protecting group (such as BOC, Fmoc, Cbz andthe like). Alternatively, G^(5P) is a nitro group, or G^(5P)-(CH₂)_(q)—is NC—(CH₂)_(q-1)—, wherein the nitro group or the cyano group issubsequently reduced to an amino group using standard reagents andprocedures.

The reaction of compound 2 with compound 13a or 13b is typicallyconducted using the reagents and reaction conditions described hereinfor the reductive alkylation of an amine with an aldehyde or ketone(e.g., compound 1 and compound 2). Upon completion of the reaction, theproduct is typically isolated using conventional procedures, such asextraction, recrystallization, chromatography, and the like.

Compounds of formula 13a and 13b are commercially available, known inthe art or can be prepared using routine variations of procedures knownin the art.

Representative compounds of formula 13a include, by way of example,methyl 3-formylbenzoate, methyl 4-formylbenzoate,methyl(3-formylphenyl)acetate, methyl(4-formylphenyl)acetate, methyl3-(3-formylphenyl)propionate, methyl 3-(4-formylphenyl)propionate,(3-formylphenyl)carbamic acid tert-butyl ester, (4-formylphenyl)carbamicacid tert-butyl ester, (3-formylbenzyl)carbamic acid tert-butyl ester,(4-formylbenzyl)carbamic acid tert-butyl ester,[2-(3-formylphenyl)ethyl]carbamic acid tert-butyl ester,[2-(4-formylphenyl)ethyl]carbamic acid tert-butyl ester, and the like.

Representative compounds of formula 13b include, by way of example,methyl 3-(2-oxoethyl)benzoate, methyl 4-(2-oxoethyl)benzoate, methyl[3-(2-oxoethyl)phenyl]acetate, methyl [4-(2-oxoethyl)phenyl]acetate,methyl 3-[3-(2-oxoethyl)phenyl]propionate, methyl3-[4-(2-oxoethyl)phenyl]propionate,2-(3-tert-butoxycarbonylaminophenyl)-acetaldehyde,2-(4-tert-butoxycarbonylaminophenyl)-acetaldehyde,2-[3-(tert-butoxycarbonylaminomethyl)phenyl]acetaldehyde,2-[4-(tert-butoxycarbonylaminomethyl)phenyl]acetaldehyde,2-{3-[2-(tert-butoxycarbonylamino)-ethyl]phenyl}acetaldehyde,2-{4-[2-(tert-butoxycarbonylamino)ethyl]phenyl}-acetaldehyde, and thelike.

Intermediate compounds of formula 12 can also be prepared by reacting acompound of formula 14:

with a compound of formula 15:

wherein L¹ is a leaving group, such as chloro, bromo, iodo, tosyl, nosyland the like; and P⁹ is a hydroxyl-protecting group, such as benzyl andthe like. The resulting product is then partially deprotected (byremoval of P⁷ or P⁸, and P⁹) to provide a compound of formula 12.

The reaction of compound 14 with compound 15 is typically conducted byreacting compound 14 with about 0.95 to about 1.1 molar equivalents ofcompound 15 in the presence of an excess amount of a base.Representative bases include, for example, sodium bicarbonate, sodiumcarbonate, potassium bicarbonate, potassium carbonate, trialkylamines(such as triethylamine, diisopropylethylamine, etc.) and the like. Thisreaction is typically conducted at a temperature ranging from about 20°C. to about 120° C., (e.g., about 100° C.) for about 2 to about 24 hoursor until the reaction is substantially complete. Typically, thisreaction is conducted in a diluent, such as N-methyl-pyrrolidinone,acetonitrile and the like. If desired, this reaction can be facilitatedby subjecting the reaction mixture to microwave radiation (e.g., 300watts). The reaction can also be conducted neat, i.e., in the absence ofa diluent. Additionally, if desired, an excess of amine 14 can be usedinstead of another base. Upon completion of the reaction, the product istypically isolated using conventional procedures, such as extraction,recrystallization, chromatography and the like.

The resulting product is then partially deprotected (i.e., P⁷ or P⁸ isremoved; and P⁹ is removed, if desired) to provide a compound of formula12. The particular conditions used to remove the protecting groups willdepend on the particular groups employed. For example, when P⁷ is C₁₋₆alkyl, such groups are typically removed by hydrolysis of the estermoiety with a base, such as, lithium hydroxide, sodium hydroxide,potassium hydroxide and the like, in a diluent, such as a mixture ofmethanol and water and the like. This reaction is typically conducted atambient temperature for about 30 minutes to about 24 hours or until thereaction is substantially complete. When P⁸ is a tert-butoxycarbonylgroup, this group is typically removed under acidic hydrolysisconditions, such as 20% trifluoroacetic acid in DCM at ambienttemperature. When P⁹ is a benzyl group, this group is readily removed byhydrogenolysis. Typically, this reaction is conducted by contacting thecompound with hydrogen in the presence of a catalyst, such as apalladium catalyst. Representative catalysts include palladium hydroxideon carbon, palladium on carbon, and the like. Generally, thisdebenzylation reaction is conducted in the presence of an acid, such asacetic acid, formic acid and the like. This reaction is typicallyconducted at a temperature ranging from about 10° C. to about 50° C.(e.g. about 25° C.) for about 6 to about 24 hours or until the reactionis substantially complete. Typically, this reaction is conducted in adiluent, such as methanol, ethanol and the like. Upon completion of thereaction, the product can be isolated using conventional procedures,such as extraction, recrystallization, chromatography and the like, orused directly in the next reaction.

Compounds of formula 14 are known in the art or can be prepared fromcommercially available starting materials and reagents using knownprocedures. For example, compounds of formula 14 can be prepared byreacting 13a or 13b with a benzyl amine under reductive alkylationconditions and then removing the benzyl group by hydrogenolysis toafford the compound of formula 14. Representative benzyl amines that maybe used include, benzyl amine, (S)-1-phenylethylamine,(R)-1-phenylethylamine, and the like. In particular, chiral benzylamines are useful for preparing intermediates of formula 14 where R⁵ ispresent (i.e., e=1) and the carbon atom to which R⁵ is attached has aparticular stereochemistry (i.e., R or S).

Additionally, compounds of formula 14 where R⁵ and R⁶ are independentlymethyl or ethyl (i.e., not hydrogen) are known in the art or can beprepared using routine procedures as exemplified by the teachings inU.S. Patent Application Publication 2005/0171147 A1, published Aug. 4,2005; U.S. Patent Application Publication 2005/0277632 A1, publishedDec. 15, 2005; WO 2005/092861 A1, published Oct. 6, 2005; and the like.

Compounds of formula 15 are also known in the art or can be preparedfrom commercially available starting materials and reagents using knownprocedures. For example, the preparation of a compound of formula 15,where L¹ is bromo; P¹ is tert-butyldimethylsilyl, and P⁹ is benzyl isdescribed in U.S. Patent Application Publication No. 2006/0035931,published Feb. 16, 2006.

Alternatively, compounds of formula 12 are prepared by reacting acompound of formula 16:

with a compound of formula 17:

wherein L² is a leaving group, such as chloro, bromo, iodo, tosyl, nosyland the like; and P¹⁰ is an amino-protecting group, such as benzyl andthe like. In this embodiment, R⁵ and R⁶ when present, are typicallyhydrogen. This reaction is conducted under conditions similar to thosedescribed for the reaction of 14 and 15. Compounds of formula 16 and 17are known in the art or can be prepared from commercially availablestarting materials and reagents using known procedures. For example, thepreparation of a compound of formula 17, where P¹ istert-butyldimethylsilyl, P⁹ is benzyl, and P¹⁰ is benzyl is described inU.S. Patent Application Publication No. 2006/0035931, published Feb. 16,2006.

If desired, a pharmaceutically acceptable salt of a compound of formulaI is prepared by contacting the free base form of the compound offormula I with a pharmaceutically acceptable acid.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth herein.

Pharmaceutical Compositions, Combinations and Formulations

Compounds of formula I can be formulated with a carrier or excipient toform a pharmaceutical composition or formulation. Such pharmaceuticalcompositions will typically contain a therapeutically effective amountof a compound of formula I. In some cases, however, the pharmaceuticalcomposition may contain more than a therapeutically effective amount,e.g., a concentrated bulk composition; or less than a therapeuticallyeffective amount, e.g., individual unit doses designed for multipleadministrations to achieve a therapeutically effective amount.

The pharmaceutical composition will typically contain from about 0.01 toabout 95 percent by weight of a compound of formula I including, forexample, from about 0.01 to about 30 percent by weight; or from about0.01 to about 10 percent by weight; or from about 0.01 to about 1percent by weight.

Such pharmaceutical compositions are typically prepared usingconventional carriers or excipients. The choice of a particular carrieror excipient, or combinations of carriers or excipients, will depend onvarious factors such as the mode of administration for the composition,or the medical condition or disease state being treated. Many suitablecarriers and excipients for preparing pharmaceutical compositions arecommercially available. For example, such materials can be purchasedfrom Sigma (St. Louis, Mo.). Procedures and materials for preparingpharmaceutical compositions suitable for a particular mode ofadministration are described in the pharmaceutical arts including, forexample, Remington: The Science and Practice of Pharmacy, 20^(th)Edition, Lippincott Williams & White, Baltimore, Md. (2000); and H. C.Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems,7^(th) Edition, Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical composition is typically prepared by thoroughly andintimately mixing or blending a compound of formula I with apharmaceutically acceptable carrier and any optional ingredients. Ifnecessary or desired, the resulting uniformly blended mixture can thenbe shaped or loaded into tablets, capsules, pills, canisters,cartridges, dispensers and the like using conventional procedures andequipment.

In one embodiment, the pharmaceutical composition is suitable forinhaled administration. Pharmaceutical compositions for inhaledadministration will typically be in the form of an aerosol or a powder.Such compositions are generally administered using well-known deliverydevices, such as a nebulizer inhaler, a metered-dose inhaler (MDI), adry powder inhaler (DPI) or a similar delivery device.

In a particular embodiment, the pharmaceutical composition comprisingthe therapeutic agent is administered by inhalation using a nebulizerinhaler. Such nebulizer devices typically produce a stream of highvelocity air that causes the pharmaceutical composition comprising thetherapeutic agent to spray as a mist that is carried into the patient'srespiratory tract. Accordingly, when formulated for use in a nebulizerinhaler, the therapeutic agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the therapeutic agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles. Nebulizer devices suitable for administeringtherapeutic agents by inhalation are described in the art or suchdevices are commercially available. For example, representativenebulizer devices or products include the Respimat Softmist Inhalaler(Boehringer Ingelheim); the AERx Pulmonary Delivery System (AradigmCorp.); the PARI LC Plus Reusable Nebulizer (Pari GmbH); and the like.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I. In oneembodiment, the solution has a pH of about 4 to about 6.

In another particular embodiment, the pharmaceutical compositioncomprising the therapeutic agent is administered by inhalation using adry powder inhaler. Such dry powder inhalers typically administer thetherapeutic agent as a free-flowing powder that is dispersed in apatient's air-stream during inspiration. In order to achieve afree-flowing powder, the therapeutic agent is typically formulated witha suitable excipient such as lactose, starch, mannitol, dextrose,polylactic acid (PLA), polylactide-co-glycolide (PLGA) or combinationsthereof. Typically, the therapeutic agent is micronized and combinedwith a suitable carrier to form a blend suitable for inhalation.Accordingly, in one embodiment, the compound of formula I is inmicronized form.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry milled lactose and micronized particles of acompound of formula I.

Such a dry powder formulation can be made, for example, by combining thelactose with the therapeutic agent and then dry blending the components.Alternatively, if desired, the therapeutic agent can be formulatedwithout an excipient. The pharmaceutical composition is then typicallyloaded into a dry powder dispenser, or into inhalation cartridges orcapsules for use with a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are described in the art or suchdevices are commercially available. For example, representative drypowder inhaler delivery devices or products include Aeolizer (Novartis);Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler(GlaxoSmithKline); Diskus/Accuhaler (GlaxoSmithKline); Easyhaler (OrionPharma); Eclipse (Aventis); FlowCaps (Hovione); Handihaler (BoehringerIngelheim); Pulvinal (Chiesi); Rotahaler (GlaxoSmithKline);SkyeHaler/Certihaler (SkyePharma); Twisthaler (Schering-Plough);Turbuhaler (AstraZeneca); Ultrahaler (Aventis); and the like.

In yet another particular embodiment, the pharmaceutical compositioncomprising the therapeutic agent is administered by inhalation using ametered-dose inhaler. Such metered-dose inhalers typically discharge ameasured amount of the therapeutic agent using a compressed propellantgas. Accordingly, pharmaceutical compositions administered using ametered-dose inhaler typically comprise a solution or suspension of thetherapeutic agent in a liquefied propellant. Any suitable liquefiedpropellant may be employed including hydrofluoroalkanes (HFAs), such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and chlorofluorocarbons,such as CCl₃F. In a particular embodiment, the propellant ishydrofluoroalkanes. In some embodiments, the hydrofluoroalkaneformulation contains a co-solvent, such as ethanol or pentane, and/or asurfactant, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof formula 1; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing thetherapeutic agent, ethanol (if present) and the surfactant (if present).To prepare a suspension, the therapeutic agent is micronized and thencombined with the propellant. The formulation is then loaded into anaerosol canister, which typically forms a portion of a metered-doseinhaler device.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are described in the art or such devices arecommercially available. For example, representative metered-dose inhalerdevices or products include AeroBid Inhaler System (ForestPharmaceuticals); Atrovent Inhalation Aerosol (Boehringer Ingelheim);Flovent (GlaxoSmithKline); Maxair Inhaler (3M); Proventil Inhaler(Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); and the like.

In another embodiment, the pharmaceutical composition is suitable fororal administration. Pharmaceutical compositions for oral administrationmay be in the form of capsules, tablets, pills, lozenges, cachets,dragees, powders, granules; or as a solution or a suspension in anaqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilliquid emulsion; or as an elixir or syrup; and the like; each containinga predetermined amount of a compound of the embodiments as an activeingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionwill typically comprise a compound of formula I and one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical composition. Examples of pharmaceuticallyacceptable antioxidants include: (1) water-soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfate sodium sulfite and the like; (2) oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and (3) metal-chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP),hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylicacid ester copolymers, cellulose acetate trimellitate (CAT),carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl celluloseacetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical composition may also be formulated toprovide slow or controlled release of the active ingredient using, byway of example, hydroxypropyl methyl cellulose in varying proportions;or other polymer matrices, liposomes and/or microspheres.

Additionally, the pharmaceutical composition may optionally contain anopacifying agent and may be formulated so that the active ingredientreleased primarily in a certain portion of the gastrointestinal tract orin a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. The active ingredient or apharmaceutical composition containing the active ingredients can also bein micro-encapsulated form.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionmay be packaged in a unit dosage form. The term “unit dosage form” meansa physically discrete unit suitable for dosing a patient, i.e., eachunit containing a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such a unit dosage formmay be a capsule, tablet, pill, and the like.

The compounds of formula I can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, acompound of the embodiments can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

Additionally, a compound of formula I can be administered parenterally,i.e., intravenously, subcutaneously or intramuscularly. For parenteraladministration, a compound of formula I is typically dissolved in acarrier acceptable for parenteral administration, such as sterile water,saline, vegetable oil and the like. By way of illustration, anintravenous composition typically comprises a sterile aqueous solutionof a compound of formula I, wherein the solution has a pH in the rangeof about 4 to about 7.

If desired, a compound of formula I may be administered in combinationwith one or more other therapeutic agents. In this aspect of theinvention, a compound of formula I is either physically mixed with theother therapeutic agent to form a composition containing both agents; oreach agent is present in separate and distinct compositions which areadministered to the patient simultaneously or sequentially.

For example, a compound of formula I can be combined with secondtherapeutic agent using conventional procedures and equipment to form acomposition comprising a compound of formula I and a second therapeuticagent. Additionally, the therapeutic agents may be combined with apharmaceutically acceptable carrier to form a pharmaceutical compositioncomprising a compound of formula I, a second therapeutic agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.

Alternatively, the therapeutic agents may remain separate and distinctbefore administration to the patient. In this embodiment, thetherapeutic agents are not physically mixed together beforeadministration but are administered simultaneously or sequentially asseparate compositions. For example, a compound of formula I can beadministered by inhalation simultaneously or sequentially with anothertherapeutic agent using an inhalation delivery device that employsseparate compartments (e.g. blister packs) for each therapeutic agent.Alternatively, the combination may be administered using separatedelivery devices, i.e., one delivery device for each therapeutic agent.Additionally, the therapeutic agents can be delivered by differentroutes of administration, i.e., one by inhalation and the other by oraladministration.

Any therapeutic agent compatible with the compounds of formula I may beused in combination with such compounds. In a particular embodiment, thesecond therapeutic agent is one that is effectively administered byinhalation. By way of illustration, representative types of therapeuticagents that may be used with the compounds of the embodiments include,but are not limited to, anti-inflammatory agents, such as steroidalanti-inflammatory agents (including corticosteroids andglucocorticoids), non-steroidal anti-inflammatory agents (NSAIDs), andPDE₄ inhibitors; bronchodilators, such as PDE₃ inhibitors, adenosine 2bmodulators and β₂ adrenergic receptor agonists; antiinfective agents,such as Gram-positive antibiotics, Gram-negative antibiotics, andantiviral agents; antihistamines; protease inhibitors; afferentblockers, such as D₂ agonists and neurokinin modulators; and muscarinicreceptor antagonists (antichlolinergic agents). Numerous examples ofsuch therapeutic agents are well known in the art. Suitable doses forthe other therapeutic agents administered in combination with a compoundof the embodiments are typically in the range of about 0.05 μg/day toabout 500 mg/day.

In a particular embodiment, a compound of formula I is administered incombination with a steroidal anti-inflammatory agent. Representativeexamples of steroidal anti-inflammatory agents that can be used incombination with the compounds of the embodiments include, but are notlimited to, beclomethasone dipropionate; budesonide; butixocortpropionate;20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-11β-hydroxy-17β-(methylthio)androsta-4-en-3-one(RPR-106541); ciclesonide; dexamethasone;6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methytl-1,3-thiazole-5-carbonyl)oxy]-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-17β-carbothioicacid (S)-(2-oxotetrahydrofuran-3S-yl) ester; flunisolide; fluticasonefuroate; fluticasone propionate; methyl prednisolone; mometasonefuroate; prednisolone; prednisone; rofleponide; ST-126 ; triamcinoloneacetonide; and the like, or pharmaceutically acceptable salts orsolvates thereof. Such steroidal anti-inflammatory agents arecommercially available or can be prepared using conventional proceduresand reagents. For example, the preparation and use of steroidalanti-inflammatory agents is described in U.S. Pat. No. 6,537,983, issuedMar. 25, 2003; U.S. Pat. No. 6,750,210 B2, issued Jun. 15, 2004; U.S.Pat. No. 6,759,398 B2, issued Jul. 6, 2004; U.S. Pat. No. 6,858,596 B2,issued Feb. 22, 2005; U.S. Pat. No. 7,101,866 B2, issued Sep. 5, 2006;and the references cited therein.

When employed, the steroidal anti-inflammatory agent is typicallyadministered in an amount that produces a therapeutically beneficialeffect when co-administered with a compound of the embodiments.Typically, the steroidal anti-inflammatory agent will be administered inan amount sufficient to provide from about 0.05 μg to about 500 μg perdose.

The following examples illustrate representative pharmaceuticalcompositions:

A. Dry Powder Composition

A micronized compound of formula I (100 mg) is blended with milledlactose (25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). This blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10 μg to about 500 μg of the compoundof formula I per dose. The contents of the blisters are administeredusing a dry powder inhaler.

B. Dry Powder Composition

A micronized compound of formula I (1 g) is blended with milled lactose(200 g) to form a bulk composition having a weight ratio of compound tomilled lactose of 1:200. The blended composition is packed into a drypowder inhalation device capable of delivering between about 10 μg toabout 500 μg of the compound of formula I per dose.

C. Dry Powder Composition

A micronized compound of formula I (100 mg) and a micronized steroidalanti-inflammatory agent (500 mg) are blended with milled lactose (30 g).This blended mixture is then loaded into individual blisters of apeelable blister pack in an amount sufficient to provide about 10 μg toabout 500 μg of the compound of formula I per dose. The contents of theblisters are administered using a dry powder inhaler.

D. Metered-Dose Inhaler Composition

A micronized compound of formula I (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of formula I per dose when administeredby the metered dose inhaler.

E. Nebulizer Composition

A compound of formula I (25 mg) is dissolved in citrate buffered (pH 5)isotonic saline (125 mL). The mixture is stirred and sonicated until thecompound is dissolved. The pH of the solution is checked and adjusted,if necessary, to pH 5 by slowly adding aqueous IN sodium hydroxide. Thesolution is administered using a nebulizer device that provides about 10μg to about 500 μg of the compound of formula I per dose.

F. Hard Gelatin Capsules

A compound of formula I (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is loaded into a hard gelatin capsule (500 mg of compositionper capsule) that is administered orally.

G. Injectable Composition

A compound of formula I (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Utility

The compounds of formula I possess both muscarinic receptor antagonistactivity and β₂ adrenergic receptor agonist and therefore, suchcompounds are expected to be useful as therapeutic agents for treatingmedical conditions mediated by muscarinic receptors and/or β₂ adrenergicreceptors, i.e., medical conditions that are ameliorated or moderated bytreatment with a muscarinic receptor antagonist or a β₂ adrenergicreceptor agonist. Such medical conditions are well known to those ofordinary skill in the art as exemplified by the teachings of Eglen etal., Muscarinic Receptor Subtypes: Pharmacology and TherapeuticPotential, DN&P 10(8), 462-469 (1997); Emilien et al., CurrentTherapeutic Uses and Potential of beta-Adrenoceptor Agonists andAntagonists, European J. Clinical Pharm., 53(6), 389-404 (1998); and thereferences cited therein. Such medical conditions include, by way ofexample, pulmonary disorders or diseases associated with reversibleairway obstruction, such as chronic obstructive pulmonary disease (e.g.,chronic and wheezy bronchitis and emphysema), asthma, pulmonaryfibrosis, adult/acute respiratory distress syndrome (ARDS), chronicrespiratory obstruction, bronchial hyperactivity, allergic rhinitis,pneumoconiosis (such as aluminosis, anthracosis, asbestosis, chalicosis,ptilosis, siderosis, silicosis, tabacosis and byssinosis), and otherpulmonary disorders of unknown origin which benefit from therapeuticagent-induced bronchodilation. Additionally, other conditions known tobe treatable, at least in part, with a muscarinic receptor antagonist ora β₂ adrenergic receptor agonist include premature labor, depression,congestive heart failure, skin diseases (e.g., inflammatory, allergic,psoriatic and proliferative skin diseases), conditions where loweringpeptic acidity is desirable (e.g., peptic and gastric ulceration) andmuscle wasting disease.

Accordingly, one embodiment of this invention relates to a method fortreating a pulmonary disorder, the method comprising administering to apatient in need of treatment a therapeutically effective amount of acompound of formula I. When used to treat a pulmonary disorder, thecompounds of formula I will typically be administered by inhalation inmultiple doses per day, in a single dose per day or a single dose perweek. Generally, the dose for treating a pulmonary disorder is expectedto range from about 10 μg/day to about 1500 μg/day; such as about 25μg/day to about 1000 μg/day; including about 50 μg/day to about 500μg/day.

In one of its method aspects, this invention relates to a method oftreating chronic obstructive pulmonary disease or asthma, the methodcomprising administering to a patient a therapeutically effective amountof a compound of formula I. Generally, the dose for treating COPD orasthma is expected to range from about 10 μg/day to about 1500 μg/day.In particular, this method includes alleviating the symptoms of COPD orasthma. The term “COPD” is understood by those of ordinary skill in theart to include a variety of respiratory conditions, including chronicobstructive bronchitis and emphysema, as exemplified by the teachings ofBarnes, Chronic Obstructive Pulmonary Disease, N. Engl. J. Med., 2000:343:269-78, and the references cited therein.

When administered by inhalation, the compounds of formula I typicallyhave the effect of producing bronchodilation. Accordingly, in another ofits method aspects, the invention relates to a method of producingbronchodilation in a mammal, the method comprising administering to themammal a bronchodilation-producing amount of a compound of formula I.Generally, the dose for producing bronchodilation will range from about10 μg/day to about 1500 μg/day.

When used as a therapeutic agent, the compounds of formula 1 areoptionally administered in combination with another therapeutic agent oragents. In particular, by administering the compounds of formula I witha steroidal anti-inflammatory agent, triple therapy, i.e., muscarinicreceptor antagonist activity, β₂ adrenergic receptor agonist activity,and anti-inflammatory activity, is expected using only two therapeuticagents. Since pharmaceutical compositions (and combinations) containingtwo therapeutic agents are typically easier to formulate and/oradminister compared to compositions containing three therapeutic agents,such two component compositions provide a significant advantage overcompositions containing three therapeutic agents. Accordingly, inparticular embodiments, the pharmaceutical compositions, combinationsand methods of this invention further comprise a steroidalanti-inflammatory agent.

Since compounds of formula I possess both muscarinic receptor antagonistand activity β₂ adrenergic agonist activity, such compounds are alsouseful as research tools for investigating or studying biologicalsystems or samples having muscarinic receptors or β₂ adrenergicreceptors. Additionally, such compounds are useful in screening assaysto discover, for example, new compounds having both muscarinic receptorantagonist activity and adrenergic agonist activity. The biologicalsystems or samples employed may comprise muscarinic receptors or β₂adrenergic receptors or both. Any suitable biological system or samplehaving muscarinic receptors and/or β₂ adrenergic receptors may beemployed in such studies which may be conducted either in vitro or invivo. Representative biological systems or samples suitable for suchstudies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, mammals (such as mice, rats, guineapigs, rabbits, dogs, pigs, etc.), and the like.

When used as a research tool, a biological system or sample comprising amuscarinic receptor and/or a β₂ adrenergic receptor is typicallycontacted with a muscarinic receptor-antagonizing or β₂ adrenergicreceptor-agonizing amount of a compound of formula I. The effects on thebiological system or sample produced by the compound are then determinedor measured using conventional procedures and equipment, such as bymeasuring binding in a radioligand binding assays or ligand-mediatedchanges in a functional assay or by determining the amount ofbronchoprotection provided by the compound in a bronchoprotection assayin a mammal. Representative functional assays include ligand-mediatedchanges in intracellular cyclic adenosine monophosphate (cAMP);ligand-mediated changes in activity of the enzyme adenylyl cyclase(which synthesizes cAMP); ligand-mediated changes in incorporation ofguanosine 5′-O-(thio)triphosphate ([³⁵S]GTP S) into isolated membranesvia receptor catalyzed exchange of [³⁵S]GTP S for GDP; ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.); and the like. Compounds of formula I are expected toantagonize or decrease the activation of a muscarinic receptor oragonize or cause activation of a β₂ adrenergic receptor and in thefunctional assays listed herein or in assays of a similar nature. Thecompounds of formula I will typically be used in these studies at aconcentration ranging from about 0.1 nanomolar to about 100 nanomolar.

Additionally, the compounds of formula I can be used as research toolsfor evaluating other chemical compounds. In this aspect, a compound offormula I is used as a standard in an assay to allow comparison of theresults obtained with a test compound and the compound of formula I. Forexample, muscarinic receptor and/or β₂ adrenergic receptor binding data(as determined, for example, by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe muscarinic receptor and/or β₂ adrenergic receptor binding data for acompound of formula I to identify those test compounds that havedesirable binding, i.e. test compounds having binding about equal orsuperior to a compound of formula I, if any. Alternatively, for example,bronchoprotective effects can be determined for test compounds and acompound of formula I in a bronchoprotection assay in a mammal and thisdata compared to identify test compounds providing about equal orsuperior bronchoprotective effects or duration of action. This aspectincludes, as separate embodiments, both (i) the generation of comparisondata (using the appropriate assays) and (ii) the analysis of the testdata to identify test compounds of interest.

The properties and utility of the compounds of formula I can bedemonstrated using various in vitro and in vivo assays known to those ofordinary skill in the art. For example, representative assays aredescribed in further detail in the following Examples.

EXAMPLES

The following examples are provided to illustrate various representativeembodiments and aspects of this invention and are not intended to limitthe scope of this invention in any way unless specifically indicated.

All reagents, starting materials and solvents used in the followingexamples were purchased from commercial suppliers (such as AldrichChemical Company, Milwaukee, Wis.) and were used without furtherpurification unless otherwise indicated.

¹H NMR spectra were recorded on a 400 MHz Varian AS400 spectrometer,unless otherwise indicated. Chemical shifts are reported as δ values inppm relative to tetramethylsilane (TMS) as an internal standard.Coupling constants (J values) are given in hertz (Hz) and multiplicitiesare reported using the following abbreviations: s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet, br=broad, nd=not determined.

Liquid Chromatography Mass Spectroscopy (LC-MS) Conditions

LC-MS data were obtained using an Agilent 1100 Liquid ChromatographySystem —G1312A Binary Pump (Agilent Technologies), a ZORBAX RapidResolution 3.5 μm Rx, Bonus-RP column (3.5 μm particle size; 2.1 mm×50mm) (Agilent Technologies) and API 150EX Single Quadrupole LC/MS MassSpectrometer (Perkin-Elmer Sciex Instruments). The solvent systems usedwere:

Solvent A: 98% water and 2% acetonitrile (v/v)+1 mL/L TFA

Solvent B: 90% acetonitrile and 10% water (v/v)+1 mL/L TFA

Flow Rate: 500 μL/min

Gradient: (Method 10-90): 10% B to 90% B over 3 min

-   -   (Method 2-90): 2% B to 90% B over 3 min    -   (Method 10-70): 10% B to 70% B over 3 min.        HPLC Conditions

HPLC was conducted using an HP 1100 Series HPLC System (AgilentTechnologies) and a ZORBAX Rapid Resolution 3.5 μm Rx, Bonus-RP column(3.5 μm particle size; 2.1 mm×50 mm) (Agilent Technologies) or anAscentis Express C18 HPLC column (2.7 μm particle size, 3.0 mm×3 cm).The solvent systems used were:

Solvent A: 98% water and 2% acetonitrile (v/v)+1 mL/L TFA

Solvent B: 90% acetonitrile and 10% water (v/v)+1 mL/L TFA

Flow Rate: 500 μL/min

Gradient: (Method 10-50): 10% B to 50% B over 6 min

-   -   (Method 10-70): 10% B to 70% B over 6 min    -   (Method 2-90): 2% B to 90% B over 6 min.

Example 1 Preparation of3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic Acid

A stirred solution of biphenyl-2-ylcarbamic acid piperidin-4-yl ester(50.0 g, 168.7 mmol) (see, e.g., U.S. Patent Publication No.2006/0035931 A1, published Feb. 16, 2006) and acrylic acid (15.1 mL,219.3 mmol) in DCM (500 mL) was heated at 50° C. overnight. The reactionmixture was concentrated under reduced pressure and the residue wasdissolved in MeOH (600 mL). The resulting solution was heated at 75° C.for 2 h and then allowed to stand at room temperature for about 48 h.The resulting solid was collected by filtration, washed with MeOH, anddried to give the title compound (61.5 g, 99% yield).

Example 2 Preparation of4-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methyl-amino)butyricAcid Methyl Ester

To a stirred mixture of 4-methylaminobutyric acid methyl esterhydrochloride (546 mg, 3.26 mmol) and3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid (1.20 g,3.26 mmol) in DCM (15 mL) was addedN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.36 g, 3.58 mmol) followed byN,N-diisopropylethylamine (1.42 mL, 8.15 mmol). LC-MS (Method 2-90)showed product was present (Rt 3.11 min; m/z 482.4 [M+H]⁺). Water andDCM were added and the layers were separated. The organic layer waswashed with water (2×) and brine, dried over sodium sulfate, filteredand concentrated under reduced pressure to give the title compound (2.0g, 100% yield) as a light yellow oil.

Example 3 Preparation of4-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)butyricAcid

To a stirred solution of4-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionyl}methylamino)butyricacid methyl ester (1.00 g, 2.08 mmol) in THF (10 mL) was added dropwiseaqueous sodium hydroxide (1.0 M, 10.4 mL, 10.4 mmol) and the reactionmixture was stirred overnight at room temperature. LC-MS (Method 10-90)showed product was present (Rt 3.34 min; in/z 468.2 [M+H]⁺). The pH ofthe mixture was adjusted to pH 5 with aqueous hydrochloric acid (6 M)and the mixture was concentrated under reduced pressure. Aqueousammonium chloride solution was added to the residue and this mixture waswashed with EtOAc. The pH of the aqueous layer was adjusted to pH 4 withphosphate buffer solution and the aqueous layer was then extracted witha 1:3 mixture of isopropyl acetate/chloroform (4×). The combined organiclayers were dried over sodium sulfate, filtered and concentrated underreduced pressure to give the title compound as a colorless oil, whichwas used without further purification.

Example 4 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-(1,3-Dioxolan-2-yl)-phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a solution of4-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionyl}methylamino)butyricacid (75 mg, 0.16 mmol) and 4-(1,3-dioxolan-2-yl)phenylamine (26 mg,0.16 mmol) in DCM (3 mL) was addedN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (73.2 mg, 0.192 mmol) followed byN,N-diisopropylethylamine (55.9 μL, 0.321 mmol). The reaction mixturewas stirred at room temperature for about 48 h. LC-MS (Method 2-90)showed product was present (Rt 3.87 min; m/z 615.4 [M+H]⁺). Water andDCM were added and the layers were separated. The organic layer waswashed with water (2×) and brine, dried over sodium sulfate, filteredand concentrated under reduced pressure to give the title compound as ayellow oil, which was used without further purification. In subsequentexperiments, this compound was also isolated as a filterable solid.

Example 5 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-Formylphenylcarbamoyl)-propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-(1,3-dioxolan-2-yl)phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylester (98 mg, 0.16 mmol) in acetonitrile (2 mL) was added aqueoushydrochloric acid (3 M, 1.07 mL). The resulting dark orange solution wasstirred at room temperature. Water was added and the resulting mixturewas extracted with DCM (2×). The combined organic layers were dried oversodium sulfate, filtered and concentrated under reduced pressure to givethe title compound, which was used without further purification.

Example 6 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-{[(R)-2-(tert-Butyldimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-formyl-phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylester (91 mg, 0.16 mmol) in DCM (2 mL) was added5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-oneacetic acid salt (101 mg, 0.256 mmol) (see, e.g., U.S. PatentPublication No. 2006/0035931 A1, published Feb. 16, 2006) followed byMeOH (1 mL). The resulting yellow solution was stirred at roomtemperature for 10 min and then sodium triacetoxyborohydride (89 mg,0.40 mmol) was added and this mixture was stirred overnight. LC-MS(Method 2-90) showed product was present (Rt 3.25 min; m/z 889.8[M+H]⁺). Water and DCM were added and the layers were separated. Theorganic layer was washed with water (2×) and brine, dried over sodiumsulfate, filtered and concentrated under reduced pressure to give thetitle compound, which was used without further purification.

Example 7 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-{[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-yl EsterDitrifluoroacetic Acid Salt (Compound I-1)

To a solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylester (˜0.16 mmol) in DCM (3 mL) was added triethylaminetrihydrofluoride (260 μL, 1.60 mmol). The reaction mixture was stirredat room temperature overnight. LC-MS (Method 2-90) showed product waspresent (Rt 3.00 min; m/z 775.4 [M+H]⁺). The reaction mixture wasconcentrated under reduced pressure and the residue was purified by HPLCto give the title compound (27.8 mg, 39% yield, 94% purity).

Example 8 Preparation of [3-(Tritylamino)phenyl]acetic Acid

To a stirred solution of (3-aminophenyl)acetic acid (30.20 g, 199.8mmol) in pyridine (200 mL) under nitrogen at 0° C. was added dropwise asolution of trityl bromide (77.5 g, 240 mmol) in DCM (120 mL) over aperiod of 5 min. The reaction mixture was stirred at room temperaturefor 14 h and then concentrated under reduced pressure. To the residuewas added DCM (˜800 mL) and water (˜800 mL) and the layers wereseparated. The organic layer was washed with water (3×˜500 mL), driedover sodium sulfate, filtered, and concentrated under reduced pressureto afford a brownish off-white solid (100.1 g). The solid was suspendedin EtOH (500 mL) and this mixture was heated at 60° C. for 1 h and thencooled to room temperature. The solid was collected by filtration,washed with EtOH and dried under high vacuum to give the title compound(67.2 g, 85% yield) as a white solid.

¹H NMR (DMSO-d₆) δ 7.25 (m); 7.13 (m); 6.70 (s); 6.63 (t); 6.55 (d);6.27 (d); 6.09 (m); 3.29 (br s); 3.16 (s).

Example 9 Preparation of 2-[3-(Tritylamino)phenyl]ethanol

To a stirred suspension of [3-(tritylamino)phenyl]acetic acid (30.00 g,76.24 mmol) in THF (126 mL) under nitrogen at 0° C. was added dropwise asolution of borane dimethyl sulfide complex in THF (2 M, 76.2 mL, 152mmol) over a period of 45 min while maintaining the temperature at ≦1.8°C. The resulting slightly yellow homogeneous solution was stirred atroom temperature for 18 h. Saturated aqueous sodium bicarbonate solution(˜300 mL) was added slowly (strongly effervescent upon initial addition)and the resulting mixture was stirred at room temperature overnight. Themixture was extracted with EtOAc (2×˜330 mL) and the combined organiclayers were dried over sodium sulfate, filtered, and concentrated underreduced pressure to give the title compound (25.6 g, 86% yield) as awhite solid.

¹H NMR (CDCl₃) δ 7.25 (m); 6.9 (t); 6.4 (m); 6.3 (m); 6.1 (s); 5.0 (s);3.5 (m); 2.5 (t).

Example 10 Preparation of 4-Nitrobenzenesulfonic Acid2-[3-(Tritylamino)phenyl]ethyl Ester

To a stirred solution of 2-[3-(tritylamino)phenyl]ethanol (5.00 g, 13.2mmol) and triethylenediamine (2.22 g, 19.8 mmol) in DCM (52.7 mL) undernitrogen at 0° C. was added portion-wise p-nitrobenzenesulfonyl chloride(3.50 g, 15.8 mmol) over a period of 5 min. The resulting mixturestirred at 0° C. for 40 min and then saturated aqueous sodiumbicarbonate solution (˜50 mL) was added. The mixture was stirred at roomtemperature for 10 min and then the layers were separated. The aqueouslayer was extracted with DCM (2×˜50 mL) and the combined organic layerswere dried over sodium sulfate, filtered, and concentrated under reducedpressure to give the title compound (7.44 g, 100% yield) as anorange-yellow foamy solid.

¹H NMR (CDCl₃) δ 8.20 (m); 7.70 (m); 7.30 (m); 6.70 (m); 6.30 (m); 6.10(m); 5.30 (s); 5.00 (s); 4.00 (t); 3.20 (m); 2.60 (m).

Example 11 Preparation of5-[(R)-2-{[2-(3-Aminophenyl)ethyl]benzylamino}-1-(tert-butyl-dimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

A stirred mixture of 4-nitrobenzenesulfonic acid2-[3-(tritylamino)phenyl]ethyl ester (7.44 g, 13.2 mmol);5-[(R)-2-benzylamino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(5.42 g, 10.5 mmol) (see, e.g., U.S. Patent Publication No. 2006/0035931A1, published Feb. 16, 2006) and sodium bicarbonate (3.32 g, 39.5 mmol)in acetonitrile (26.4 mL) was heated at 75° C. under nitrogen for 18 h.The mixture was cooled to room temperature and the pH was adjusted topH<2 with aqueous hydrochloric acid (1 N, ˜40 mL). The mixture wasstirred at room temperature for 1 h and then the pH of the mixture wasadjusted to pH 7-8 with saturated aqueous sodium bicarbonate solution(˜30 mL). Water (˜50 mL) and EtOAc (˜50 mL) were added and the layerswere separated (a small amount of dilute aqueous sodium chloridesolution was added to improve separation). The aqueous layer wasextracted with EtOAc (2×30 mL) and the combined organic layers weredried over sodium sulfate, filtered, and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography(30-60% of EtOAc in hexanes) to give the title compound (5.7 g, 68%yield) as a light yellow foamy solid.

¹H NMR (CDCl₃) δ 9.10 (s); 7.90 (br s); 7.40 (m); 7.20 (m); 7.10 (m);7.00 (t); 6.90 (s); 6.50 (m); 6.30 (s); 5.20 (s); 4.80 (m); 3.60 (m);2.80 (m); 2.60 (m); 0.80 (s); −0.2 (s).

Example 12 Preparation of 4-(tert-Butoxycarbonylmethylamino)butyric Acid

To a stirred mixture of 4-(methylamino)butyric acid hydrochloride (1.00g, 6.51 mmol) and triethylamine (2.72 mL, 19.5 mmol) in DCM (60 mL) atroom temperature was added di-tert-butyldicarbonate (1.56 g, 7.16 mmol).The resulting mixture was stirred for about 72 h. LC-MS showed productwas present (Rt 4.11 min; m/z 216.2 [M+H]⁺). DCM and water were addedand the pH of the aqueous layer was adjusted to pH 4.5 to 6 with aqueoushydrochloric acid (1 M). The layers were separated and the organic layerwas dried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound (1.5 g, 100% yield) as light yellowthick oil.

¹H NMR (CDCl₃) δ 3.28 (br s); 2.85 (s); 2.35 (t); 1.84 (t); 1.46 (s).

Example 13 Preparation of{3-[3-(2-{Benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]amino}ethyl)phenylcarbamoyl]-propyl}methylcarbamicAcid tert-Butyl Ester

To a stirred solution of 4-(tert-butoxycarbonylmethylamino)butyric acid(72.0 mg, 0.331 mmol);5-[(R)-2-{[2-(3-aminophenyl)ethyl]benzylamino}-1-(tert-butyl-dimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(210 mg, 0.331 mmol); 2,6-lutidine (46.5 μL, 0.398 mmol); andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (69.8 mg,0.364 mmol) in DMF (3 mL) at room temperature was added a solution of1-hydroxy-7-azabenzotriazole in DMF (0.5 M, 0.729 mL, 0.364 mmol). Theresulting mixture was stirred at room temperature overnight. LC-MS(Method 2-90) showed product was present (Rt 4.56 min; m/z 833.6[M+H]⁺). Water was added and this mixture was diluted with aqueous 10%lithium chloride solution and DCM. The layers were separated and theorganic layer was washed with aqueous 10% lithium chloride solution(2×), dried over sodium sulfate, filtered and concentrated under reducedpressure. The residue was further concentrated under high vacuum to givethe title compound (containing a minor amount of DMF), which was used inthe next reaction without further purification.

Example 14 Preparation ofN-[3-(2-{Benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]amino}ethyl)phenyl]-4-methylaminobutyramide

To a stirred solution of{3-[3-(2-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]amino}ethyl)-phenylcarbamoyl]propyl}methylcarbamic acid tert-butyl ester (276 mg,0.331 mmol) in DCM (3 mL) at room temperature was added trifluoroaceticacid (2 mL, 20 mmol) and the resulting mixture was stirred at roomtemperature overnight. LC-MS (Method 2-90) showed product was present(Rt 2.69 min; m/z 7.33.4 [M+H]⁺). The reaction mixture was concentratedunder reduced pressure and the residue was dissolved in DCM. Thissolution was washed with aqueous saturated sodium bicarbonate solution(2×), dried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound (249 mg, 100% yield) as ayellow-brown oil, which was used in the next reaction without furtherpurification.

Example 15 Preparation of Biphenyl-2-ylcarbamic Acid1-[2-({3-[3-(2-{Benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]-amino}ethyl)phenylcarbamoyl]propyl}methylcarbamoyl)ethyl]piperidin-4-ylEster

To a stirred solution of3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionic acid (127 mg,0.344 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (69.0 mg, 0.360 mmol) in DMF (5 mL) at room temperaturewas added a solution of 1-hydroxy-7-azabenzotriazole in DMF (0.5 M,0.720 mL, 0.360 mmol).N-[3-(2-{Benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]amino}ethyl)phenyl]-4-methylaminobutyramide (240 mg, 0.327 mmol) and 2,6-lutidine (114 μL,0.982 mmol) were added and the resulting mixture was stirred overnight.LC-MS (Method 2-90) showed product was present (Rt 4.13 min; m/z 1083.7[M⁺]). Water was added and this mixture was diluted with DCM. The layerswere separated and the organic layer was washed with aqueous 10% lithiumchloride solution (2×), dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel flash chromatography (0-20% MeOH in DCM with 1% TEA) to give thetitle compound (258 mg, 72% yield) as a yellow oil.

Example 16 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(3-{2-[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-ethyl}phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-[2-({3-[3-(2-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]-amino}ethyl)phenylcarbamoyl]propyl}methylcarbamoyl)ethyl]piperidin-4-ylester (258 mg, 0.238 mmol) in MeOH (3 mL) at room temperature was addeda solution of acetic acid in water (17.4 M, 41.4 μL, 0.720 mmol). Theresulting solution was purged with nitrogen (3 cycles of vacuumfollowing by dry nitrogen) and then palladium hydroxide (47 mg, 0.34mmol) was added. This mixture was again purged with nitrogen and thenwith hydrogen (5×) and then stirred at room temperature under hydrogen(balloon with submerged needle) for 6 h. LC-MS (Method 2-90) showedproduct was present (Rt 3.71 min; m/z 903.6 [M+H]⁺). The reactionmixture was filtered through a membrane filter and the filtrate wasconcentrated under reduced pressure to give the title compound, whichwas used in the next reaction without further purification.

Example 17 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(3-{2-[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]ethyl}phenylcarbamoyl)-propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster Ditrifluoroacetic Acid Salt (Compound I-7)

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(3-{2-[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-ethyl}phenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylester (202 mg, 0.224 mmol in DCM (4 mL) at room temperature was addedtriethylamine trihydrofluoride (364 μL, 2.24 mmol) and the resultingmixture was stirred overnight. LC-MS (Method 2-90) showed product waspresent (Rt 2.77 min; m/z 789.6 [M+H]⁺). The reaction mixture wasconcentrated under reduced pressure and the residue was purified by HPLCto give the title compound (101.6 mg, 47% yield, 100% purity).

Example 18 Preparation of Methyl(3-Bromophenyl)acetate

A stirred solution of (3-bromophenyl)acetic acid (10.0 g, 0.0465 mol)and concentrated sulfuric acid (4.5 mL) in MeOH (230 mL) was heated at40° C. for 16 h. The reaction mixture was concentrated under reducedpressure and the residue was mixed with water (50 mL) and DCM (100 mL).The layers were separated and the organic layer was concentrated underreduced pressure. The residue was purified by silica gel flashchromatography (0-10% EtOAc/hexanes) to give the title compound (19.8 g,91% yield) as a colorless oil.

LC-MS (Method 10-90): Rt 3.36 min; m/z 300.4 [M+H]⁺; 229.6 [M⁺]. ¹H NMR(CD₃OD) δ 7.48 (s, 1H); 7.44 (d, 1H); 7.26-7.25 (m, 2H); 3.71 (s, 3H);3.66 (s, 2H).

Example 19 Preparation of Methyl [3-(2-Oxopropyl)phenyl]acetate

A stirred mixture of methyl(3-bromophenyl)acetate (19.7 g, 86.0 mmol),tributyltin methoxide (37.1 mL, 129 mmol), isopropenyl acetate (14.2 mL,129 mmol), palladium acetate (961 mg, 4.28 mmol), andtri-o-tolylphosphine (2.63 g, 8.64 mmol) in toluene (70 mL) was heatedat 100° C. for 6 h. Aqueous potassium fluoride solution (4 M, 120 mL)and EtOAc (200 mL) were added and the resulting mixture was stirredovernight. The mixture was then filtered through Celite and the layerswere separated. The organic layer was washed with water and thenconcentrated under reduced pressure. The residue was purified by silicagel flash chromatography (0-8% MeOH in DCM) to give the title compound(11.8 g, 66% yield).

LC-MS (Method 10-90): Rt 4.24 min; m/z 207.3 [M+H]⁺.

Example 20 Preparation of Methyl{3-[(S)-2-((S)-1-Phenylethylamino)propyl]phenyl}acetate HydrochlorideSalt

A mixture of methyl [3-(2-oxopropyl)phenyl]acetate (2.31 g, 11.2 mmol),(S)-1-phenylethylamine (3.4 mL, 27.0 mmol), sodium triacetoxyborohydride(14 g, 68.0 mmol), and magnesium sulfate (3.2 g, 27.0 mmol) in DCM (69mL) was stirred overnight. The reaction mixture was cooled to 0° C. andsaturated aqueous sodium bicarbonate solution (150 mL) was added untileffervescence stopped. The layers were separated and the aqueous layerwas extracted with DCM (70 mL). The combined organic layers were driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was purified by silica gel flash chromatography (0-10% MeOHin DCM). Aqueous hydrochloric acid (6 N, 2 mL) was added to the productto form the hydrochloride salt. MeOH (50 mL) was added and the resultingmixture was concentrated under reduced pressure. This procedure wasrepeated and then a minimum amount of MeOH (5 mL) was added tocompletely dissolve the solid. Diisopropyl ether (9 mL) was added andthe resulting mixture was left standing at room temperature for 2 h. Theresulting precipitate was collected by filtration and washed with etherto give the title compound (2.85 g; 81% yield; 10:1 de of S,S) as awhite solid.

LC-MS (Method 2-90): Rt 2.28 min; m/z 312.2 [M+H]⁺. ¹H NMR (CD₃OD) δ7.71-7.69 (m, 4H); 7.45 (t, 1H); 7.35 (t, 1H); 7.20-7.17 (m, 3H);4.80-4.76 (m, 1H); 3.85 (s, 3H); 3.81 (s, 2H); 3.42-3.40 (m, 1H); 2.80(t, 2H); 1.86 (d, 3H); 1.37 (d, 3H).

Example 21 Preparation of Methyl [3-((S)-2-Aminopropyl)phenyl]acetate

A stirred solution of methyl{3-[(S)-2-((S)-1-phenylethylamino)propyl]-phenyl}acetate hydrochloridesalt (1.12 g, 3.60 mmol), ammonium formate (1.16 g, 257 mmol), andpalladium hydroxide (0.30 g, 2.1 mmol) in EtOH (40 mL) was heated at 75°C. for 1 h. The reaction mixture was filtered through Celite and thefilter cake was washed with EtOAc (2×50 mL). The filtrate wasconcentrated under reduced pressure and the residue was dissolved in DCM(50 mL) and 20% aqueous ammonia solution (50 mL). The layers wereseparated and the aqueous layer was extracted with DCM. The combinedorganic layers were dried over sodium sulfate, filtered and concentratedunder reduced pressure to give the title compound (437 mg, 58% yield) asa colorless oil.

LC-MS (Method 2-90): Rt 1.18 min; m/z 208.4[M+H]⁺. ¹H NMR (CD₃OD) δ 7.43(t, 1H); 7.29-7.25 (m, 3H); 3.85 (s, 3H); 3.81 (s, 2H); 3.31-3.23 (m,1H); 2.85-2.75 (m, 2H); 1.26 (d, 3H).

Example 22 Preparation of(3-{(S)-2-[(R)-2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)aceticAcid Methyl Ester

A solution of [3-((S)-2-aminopropyl)phenyl]acetic acid methyl ester (632mg, 3.05 mmol) and triethylamine (1.27 mL, 9.15 mmol) inN-methylpyrrolidinone (3.4 mL, 35 mmol) was added to8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)-ethyl]-1H-quinolin-2-one(1.5 g, 3.0 mmol) (see, e.g., U.S. Patent Publication No. 2006/0035931A1, published Feb. 16, 2006). The resulting mixture was microwaved (300watts) at 100° C. for 1.5 h. The mixture was then diluted with EtOAc (20mL) and this solution was washed repeatedly with brine (100 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was purified by silica gel flash chromatography (0-10% MeOHin DCM) to give the title compound (400 mg, 20% yield) as a yellow oil.

LC-MS (Method 2-90): Rt 2.79 min; m/z 615.4[M+H]⁺.

Example 23 Preparation of(3-{(S)-2-[(R)-2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)aceticAcid

To a solution of(3-{(S)-2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)aceticacid methyl ester (810 mg, 1.3 mmol) in a 3:2:1 mixture ofTHF/MeOH/water (4 mL) was added lithium hydroxide (158 mg, 6.59 mmol).The resulting mixture was stirred at room temperature for 3 hours andthen acidified to pH 6 with concentrated aqueous hydrochloric acid. Thismixture was concentrated under reduced pressure to give a yellow solid(307 mg, 39% yield), which was used in the next reaction without furtherpurification.

LC-MS (Method 2-90): Rt 3.10 min; m/z 601.4[M+H]⁺.

Example 24 Preparation of(3-{(S)-2-[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}phenyl)aceticAcid

A solution of(3-{(S)-2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)aceticacid (˜1.3 mmol) in EtOH (8 mL) was purged with dry nitrogen and thenpalladium on carbon (10%, ˜50% water, Degussa type, 300 mg) was added.This mixture was purged with hydrogen and then stirred under anatmosphere of hydrogen (balloon) for 1 h. The mixture was then filteredthrough Celite and the filter bed washed with MeOH (20 mL) and EtOAc (20mL). The filtrate was concentrated under reduced pressure and theresidue was purified by silica gel flash chromatography (0-20% MeOH inDCM) to give the title compound (300 mg, 40% yield) as a yellow solid.

LC-MS (Method 2-90): Rt 2.67 min; m/z 511.6 [M+H]⁺.

Example 25 Preparation of[3-(9H-Fluoren-9-ylmethoxycarbonylamino)propyl]methylcarbamic Acidtert-Butyl Ester

To a stirred solution of N-(3-aminopropyl)-N-methylcarbamic acidtert-butyl ester (2.00 g, 10.6 mmol) and sodium carbonate (2.81 g, 26.6mmol) in water (7 mL) at 0° C. was added a solution of 9-fluorenylmethylchloroformate (2.75 g, 10.6 mmol) in 1,4-dioxane (5 mL). The resultingmixture was stirred at room temperature for 2 h and then poured intowater (10 mL). This mixture was extracted with diethyl ether (2×50 mL)and the combined organic layers were dried over sodium sulfate, filteredand concentrated under reduced pressure to afford a clear oil. The oilwas purified by silica gel flash chromatography (0-70% EtOAc in hexanes)to give the title compound (4.24 g, 77% yield) as a clear oil (productcontained a minor impurity, but was used without further purification).

LC-MS (Method 2-90): Rt 4.05 min; m/z 411.2[M+H]⁺. ¹H NMR (CD₃OD) δ 7.96(d, 2H); 7.81 (d, 2H), 7.56 (t, 2H); 7.48 (t, 2H); 4.55 (d, 2H); 4.37(t, 1H); 3.41 (t, 2H); 3.27 (t, 2H); 3.01 (s, 3H); 1.89-1.85 (m, 2H);1.61 (s, 9H).

Example 26 Preparation of (3-Methylaminopropyl)carbamic Acid9H-Fluoren-9-ylmethyl Ester Hydrochloride Salt

To a stirred solution of[3-(9H-fluoren-9-ylmethoxycarbonylamino)propyl]-methylcarbamic acidtert-butyl ester (4.24 g, 10.3 mmol) in EtOAc (8 mL) was slowly addedconcentrated hydrochloric acid (2 mL, 60 mmol) and the resulting mixturewas stirred at room temperature overnight. The mixture was concentratedunder reduced pressure to give the title compound (3.21 g, 78% yield) asa white solid.

LC-MS (Method 2-90): Rt 2.32 min; m/z 311.4[M+H]⁺. ¹H NMR (CD₃OD) δ 7.82(d, 2H); 7.66 (d, 2H), 7.42 (t, 2H); 7.34 (t, 2H); 4.45 (d, 2H); 4.12(t, 1H); 3.23 (t, 2H); 2.99 (t, 2H); 2.71 (s, 3H); 1.88-1.83 (m, 2H).

Example 27 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(9H-Fluoren-9-ylmethoxycarbonylamino)propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred mixture of3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionic acid (2.8 g,7.6 mmol) and N,N-diisopropylethylamine (2.6 mL, 15 mmol) in DMF (31 mL)was added 2-chloro-1-methylpyridinium iodide (3.9 g, 15 mmol). Once thesolids had dissolved, (3-methylaminopropyl)carbamic acid9H-fluoren-9-ylmethyl ester hydrochloride salt (2.63 g, 7.58 mmol) wasadded and the resulting mixture was stirred at room temperature untilessentially complete by HPLC and LCMS. The mixture was then concentratedunder reduced pressure to remove most of the DMF and the residue wasdissolved in DCM (30 mL). This mixture was washed with water (3×20 mL)and brine (3×20 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel flash chromatography (0-20% MeOH in DCM) to give the title compound(5.0 g, 97% yield) as a white solid.

LC-MS (Method 2-90): Rt 3.24 min; m/z 661.4 [M+H]⁺.

Example 28 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(3-Aminopropyl)-methylcarbamoyl]ethyl}piperidin-4-yl Ester

To biphenyl-2-ylcarbamic acid1-(2-{[3-(9H-fluoren-9-ylmethoxycarbonyl-amino)propyl]methylcarbamoyl}ethyl)piperidin-4-ylester (2.2 g, 3.3 mmol) was added a 10% solution of piperidine (0.31 g,3.3 mmol) in DCM (3.2 mL) and the resulting mixture was shaken at roomtemperature for 1 h. The mixture was concentrated under reduced pressureand the residue was dissolved in DCM (100 mL). This mixture was washedwith water (2×20 mL) and then extracted with ammonium chloride (1 N,2×20 mL). The layers were separated and DCM was added to the aqueouslayer. The aqueous layer was made basic by addition of aqueous potassiumhydroxide (1 N) and the layers were separated. The organic layer waswashed with aqueous potassium hydroxide (1 N, 2×20 mL) and brine (1×20mL), dried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound (1.0 g, 66% yield) as a clear oil.

LC-MS (Method 2-90): Rt 1.85 min; m/z 439.4 [M+H]⁺.

Example 29 Preparation of Biphenyl-2-ylcarbamic Acid1-[2-({3-[2-(3-{(S)-2-[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}phenyl)acetylamino]propyl}methylcarbamoyl)ethyl]piperidin-4-ylEster

A mixture of(3-{(S)-2-[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}phenyl)aceticacid (135 mg, 0.264 mmol); 2-chloropyridium triflate on Wang resin(polymer-supported Mukaiyama reagent) (1.19 mmol/g loading; 635 mg,0.775 mmol) and N,N-diisopropylethylamine (132 μL, 0.755 mmol) in DMF(4.78 mL) was stirred at room temperature for 30 min.Biphenyl-2-ylcarbamic acid1-{2-[(3-aminopropyl)methylcarbamoyl]ethyl}piperidin-4-yl ester (122 mg,0.278 mmol) was added and this mixture was stirred at room temperatureovernight. The mixture was filtered and the resin was washed with DCM (4mL), MeOH (4 mL) and THF (4 mL). The filtrate was concentrated underreduced pressure and the residue was dissolved in DCM. This solution waswashed with water (2×10 mL), saturated aqueous sodium bicarbonate (2×10mL) and brine (1×10 mL); and then dried over sodium sulfate, filteredand concentrated under reduced pressure. The residue was purified bysilica gel flash chromatography (0-20% MeOH in DCM) to give the titlecompound (59 mg, 24% yield) as a white solid.

LC-MS (Method 2-90): Rt 4.35 min; m/z 931.7 [M+H]⁺.

Alternatively, this reaction can be conducted using a combination of EDCand HOBt as the coupling reagents.

Example 30 Preparation of Biphenyl-2-ylcarbamic Acid1-[2-({3-[2-(3-{(S)-2-[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}phenyl)acetylamino]-propyl}methylcarbamoyl)ethyl]piperidin-4-ylEster Ditrifluoroacetic Acid Salt (Compound I-13)

A solution of biphenyl-2-ylcarbamic acid1-[2-({3-[2-(3-{(S)-2-[(R)-2-(tert-butyl-dimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}-phenyl)acetylamino]propyl}methylcarbamoyl)ethyl]piperidin-4-ylester (59 mg, 0.063 mmol) and triethylamine trihydrofluoride (10.3 μL,0.0634 mmol) in DCM (1 mL) was microwaved (300 watts) at 80° C. for 20min. The reaction mixture was then concentrated under reduced pressureand the residue was purified by HPLC (Method 10-50) to give the titlecompound (35.2 mg, 52% yield).

LC-MS (Method 2-90): Rt 2.16 min; m/z 817.6[M+H]⁺.

Example 31 Preparation of Methyl 5-Methylaminopentanoate Hydrochloride

A stirred solution of 1-methyl-2-piperidinone (4.40 mL, 40.0 mmol) inaqueous sodium hydroxide (4 M, 11.0 mL, 44.0 mmol) was heated at 100° C.for 15 h. The reaction mixture was cooled to room temperature and thenacidified to pH 2 with concentrated hydrochloric acid. The mixture wasthen concentrated under reduced pressure to give crude5-methylaminopentanoic acid as a pinkish white solid. To the crude5-methylaminopentanoic acid was added MeOH (40.0 mL, 987 mmol) andconcentrated hydrochloric acid (0.33 mL, 4.0 mmol). The resulting cloudysolution was heated at 60° C. for 39 h at which time LC-MS showedremaining starting material. Additional concentrated hydrochloric acid(0.33 mL, 4.0 mmol) was added and the resulting mixture was heated at60° C. for 33 h and then at 65° C. for an additional 24 h. LC-MS showedremaining starting material. The reaction mixture was concentrated underreduced pressure and a solution of hydrogen chloride in MeOH (1.25 M)was added to the residue. The resulting mixture was heated at 60° C. for72 h at which time no remaining starting material was observed by LC-MS.The reaction mixture was partially concentrated under reduced pressureand the solid material that formed was removed by filtration, washingwith MeOH. The filtrate was then concentrated under reduced pressure toprovide methyl 5-methylaminopentanoate hydrochloride (7.57 g, 100%yield) as a light yellow solid.

LC-MS (Method 2-90): Rt 1.10 min; m/z 146.4 [M+H]⁺. ¹H NMR (CD₃OD) δ4.86 (s), 3.66 (s), 3.30 (t), 3.00 (t), 2.69 (s), 2.41 (t), 1.71 (m).

Example 32 Preparation of5-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)pentanoicAcid Methyl Ester

A mixture of methyl 5-methylaminopentanoate hydrochloride (7.27 g, 40.0mmol), 3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid(13.3 g, 36.0 mmol) and 1-hydroxy-7-azabenzotriazole (5.14 g, 37.8 mmol)in DCM (160 mL) and 2,6-lutidine (12.5 mL, 108 mmol) was stirred at roomtemperature for 3 h. N-(3-Dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (10.4 g, 54.0 mmol) was added and the resulting mixturewas stirred at room temperature for 2 h. A saturated aqueous sodiumbicarbonate solution (˜100 mL) was added and the layers were separated.The aqueous layer was extracted with DCM (50 mL) and the organic layerswere combined, dried over sodium sulfate, filtered and concentratedunder reduced pressure. The residue was purified by silica gel flashchromatography (30-100% EtOAc in hexanes; then 2-10% MeOH in DCM) togive the title compound (12.38 g, 69% yield) as a light yellow thickoil/white solid.

LC-MS (Method 2-90): Rt 2.43 min; m/z 496.6 [M+H]⁺. ¹H NMR (CDCl₃) δ8.10 (d, 1H), 7.40 (m, 6H), 7.20 (m, 2H), 6.58 (s, 1H), 4.74 (m, 1H),3.66 (d, 3H), 3.37 (t, 1H), 3.29 (m, 1H), 2.97 (s, 2H), 2.91 (s, 1H),2.70 (m, 4H), 2.49 (m, 2H), 2.34 (m, 4H), 1.92 (m, 2H), 1.60 (m, 5H).

Example 33 Preparation of5-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)pentanoicAcid

To a mixture of5-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}-methylamino)pentanoicacid methyl ester (10.21 g, 20.60 mmol), tert-butyl alcohol (20 mL) andwater (20 mL) was added a 1:1 mixture of LiOH:water (1.97 g, 41.2 mmol).The resulting mixture was stirred at room temperature for 4 h and thenthe pH of the mixture was adjusted to about pH 2 using aqueoushydrochloric acid (1 N). The aqueous layer was extracted with DCM (2×˜80mL) and the organic layers were combined, dried over sodium sulfate,filtered, and concentrated under reduced pressure to give the titlecompound (12.23 g, quantitative) as an off-white foamy solid (containingresidual tert-butyl alcohol).

LC-MS (Method 2-90): Rt 2.32 min; m/z 482.4 [M+H]⁺.

Example 34 Preparation of 2-(4-Nitrophenyl)-1,3-dioxolane

A stirred solution of p-nitrobenzaldehyde (101.5 g, 672 mmol), ethyleneglycol (112 mL) and p-toluenesulfonic acid (12.8 g, 67.2 mmol) intoluene (800 mL) was heated in flask equipped with a Dean-Stark trap at120° C. for 4 h. After cooling to room temperature, the reaction mixturewas concentrated under reduced pressure. To the residue was addedsaturated aqueous sodium bicarbonate (800 mL) and this mixture wasstirred at room temperature for 15 min. The resulting solid was isolatedby filtration and dried under vacuum to give the title compound (121.8g, 92% yield) as a yellow solid.

¹H NMR (DMSO-d₆): δ=8.12 (d, 2H), 7.59 (d, 2H), 5.78 (s, 1H), 3.8-4.0(m, 4H).

Example 35 Preparation of 4-(1,3-Dioxolan-2-yl)phenylamine

To a mixture of platinum dioxide (227 mg, 1.00 mmol) and sodiumbicarbonate (420 mg, 5.00 mmol) under dry nitrogen was added a solutionof 2-(4-nitrophenyl)-1,3-dioxolane (976 mg, 5.00 mmol) in EtOH (30.0mL). The reaction mixture was bubbled with hydrogen for 15 min and thenstirred under a hydrogen atmosphere (balloon) for 2 h. The reactionmixture was then filtered through a pad of Celite washing with MeOH. Thefiltrate was concentrated under reduced pressure to give the titlecompound (0.80 g, 96% yield).

Example 36 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-(1,3-Dioxolan-2-yl)-phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of5-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionyl}methylamino)pentanoicacid (2.33 g, 4.84 mmol), 4-(1,3-dioxolan-2-yl)phenylamine (800 mg, 5mmol) and N,N-diisopropylethylamine (1.26 mL, 7.26 mmol) in DCM (48.4mL) was added 1-hydroxy-7-azabenzotriazole (692 mg, 5.08 mmol) andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.39 g,7.26 mmol). The resulting mixture was stirred at room temperatureovernight. The reaction mixture was then washed with saturated aqueoussodium bicarbonate solution, dried over sodium sulfate, filtered andconcentrated under reduced pressure to give the title compound (3.04 g,100% yield) as a yellow solid.

LC-MS (Method 10-70): Rt 2.67 min; m/z 629.6 [M+H]⁺.

Example 37 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Formylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

A stirred mixture of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-(1,3-dioxolan-2-yl)phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (3.04 g, 4.84 mmol) in aqueous hydrochloric acid (1 M, 10 mL) andacetonitrile (10 mL) was heated at 50° C. for 2 h. The reaction mixturewas concentrated under reduced pressure and saturated aqueous sodiumbicarbonate solution and DCM were added to the residue. The layers wereseparated and the organic layer was dried over sodium sulfate, filteredand concentrated under reduced pressure to give the title compound (2.83g, 100% yield).

LC-MS (Method 10-70): Rt 2.67 min; m/z 585.4 [M+H]⁺.

Example 38 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-{[(R)-2-(tert-butyldimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-formyl-phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (2.83 g, 4.84 mmol) and5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-oneacetic acid salt (1.91 g, 4.84 mmol) in a 1:1 mixture of MeOH:DCM (40.0mL, 312 mmol) was added sodium triacetoxyborohydride (3.08 g, 14.5mmol). The reaction mixture was stirred at room temperature for 2 h andthen the layers were separated. The organic layer was washed withsaturated aqueous sodium bicarbonate solution, dried over sodiumsulfate, filtered and concentrated under reduced pressure to give ayellow solid. The solid was purified by silica gel flash chromatography(0-30% MeOH in DCM+0.5% NH₄OH) to give the title compound (3.60 g, 82%yield) as a yellow solid.

LC-MS (Method 10-70): Rt 2.72 min; m/z 903.8 [M+H]⁺.

Example 39 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}phenylcarbamoyl)-butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster Ditrifluoroacetic Acid Salt (Compound I-14)

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-methyl}phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (3.60 g, 3.98 mmol) in a 9:1 mixture of DCM:DMF (32.9 mL) wasadded triethylamine trihydrofluoride (1.95 mL, 12.0 mmol). The resultingmixture was stirred at room temperature overnight and then concentratedunder reduced pressure. The residue was purified by HPLC (Method 10-70)to give the title compound (1.90 g, 46% yield) as a white solid.

LC-MS (Method 10-70): Rt 2.12 min; m/z 789.6 [M+H]⁺.

Example 40 Preparation of Dibenzyl-(4-iodo-2,5-dimethylphenyl)amine

A mixture of 2,5-dimethyl-4-iodoaniline (6.44 g, 26.1 mmol), benzylbromide (11.50 g, 67.24 mmol) and potassium carbonate (8.20 g, 59.3mmol) in EtOH (100 mL) was stirred at 50° C. for 12 h. The reactionmixture was then concentrated under reduced pressure. The residue (apurple solid) was mixed with DCM and this mixture was filtered usingvacuum. The filtrate was purified by silica gel flash chromatography(0-5% MeOH in DCM) to give the title compound (9.35 g, 83% yield) as anoil.

LC-MS (Method 10-90): Rt 3.99 min; m/z 428.4 [M+H]⁺.

Example 41 Preparation of 4-Dibenzylamino-2,5-dimethylbenzaldehyde

To a stirred solution of dibenzyl-(4-iodo-2,5-dimethylphenyl)amine (9.35g, 21.9 mmol) in toluene (100 mL) under nitrogen at −15° C. was addedn-butyllithium in hexanes (1.6 M, 20.5 mL, 32.8 mmol) dropwise viasyringe over a 30 min period. The resulting mixture was stirred at −15°C. for 15 min and then N,N-dimethylformamide (1.86 mL, 24.1 mmol) wasadded dropwise over a 10 min period. After 2 h, aqueous hydrochloricacid (1 M, 46.6 mL) and brine (30 mL) were added and the resultingmixture was stirred for 15 min. The layers were separated and theorganic layer was extracted with brine, dried over magnesium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by silica gel flash chromatography (0-50% DCM in hexanes) togive the title compound (3.60 g, 49% yield) as a clear oil.

LC-MS (Method 10-90): Rt 3.76 min; m/z 330.4 [M+H]⁺.

Example 42 Preparation ofDibenzyl-[4-(1,3-dioxolan-2-yl)-2,5-dimethylphenyl]amine

To a stirred solution of 4-dibenzylamino-2,5-dimethylbenzaldehyde (3.60g, 10.9 mmol) in toluene (35.0 mL) under nitrogen was added1,2-ethanediol (1.83 mL, 32.8 mmol) and p-toluenesulfonic acid (451 mg,2.62 mmol). The reaction mixture was heated at 80° C. overnight. TLC(3:1 hexanes:EtOAc) showed a ˜50:50 mixture of product and startingmaterial. Magnesium sulfate (1.32 g, 10.9 mmol) was added and stirringwas continued for 6 h. The reaction mixture was then filtered andconcentrated under reduced pressure to give title compound (3.19 g, 78%yield) as an oil.

Example 43 Preparation of (4-Amino-2,5-dimethylphenyl)methanol

A stirred solution ofdibenzyl-[4-(1,3-dioxolan-2-yl)-2,5-dimethylphenyl)amine (2.00 g, 5.35mmol) in EtOH (14.0 mL) and EtOAc (7.00 mL) was flushed with nitrogenfor 3 min and then sodium bicarbonate (0.200 g, 2.38 mmol) and palladiumon activated carbon (10 wt. %, ˜50% water; 0.800 g, 0.360 mmol) wereadded. The reaction mixture was flushed with hydrogen gas for 3 min andthen stirred under a hydrogen atmosphere (balloon) for 3 h. The mixturewas then filtered through a pad of Celite and the pad washed with MeOH(10 mL). The filtrate was concentrated under reduced pressure to givethe title compound, which was used without any further purification.

Example 44 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl)propyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

A solution of5-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}-methylamino)pentanoicacid (1.19 g, 2.46 mmol); (4-amino-2,5-dimethylphenyl)-methanol (372 mg,2.46 mmol) and N,N-diisopropylethylamine (858 μL, 4.93 mmol) in DCM(18.5 mL) was stirred at room temperature for 30 min.N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (708 mg,3.69 mmol) was added and the resulting mixture was stirred at roomtemperature for 3 h. Saturated aqueous sodium bicarbonate solution (5mL) was added and this mixture was extracted with DCM (2×2 mL). Thecombined organic layers were dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel flash chromatography (0-10% MeOH in DCM) to give the title compound(460 mg, 31% yield) as a yellow oil.

LC-MS (Method 10-90): Rt 2.53 min; m/z 599.4 [M+H]⁺.

Example 45 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Formyl-2,5-dimethyl-phenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-hydroxymethyl-2,5-dimethylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (0.460 g, 0.748 mmol) and dimethyl sulfoxide (0.531 mL, 7.48 mmol)in DCM (2.58 mL) at 0° C. was added N,N-diisopropylethylamine (0.65 mL,3.7 mmol). Sulfur trioxide —pyridine complex (0.357 g, 2.24 mmol) wasadded and the resulting mixture was stirred at 0° C. for 1 h. Water (3mL) was added and the layers were separated. The organic layer was driedover sodium sulfate and filtered to give the title compound in a DCMsolution, which was used immediately in the next reaction.

LC-MS (Method 10-90): Rt 2.44 min; m/z 613.4 [M+H]⁺.

Example 46 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-[{(R)-2-(tert-butyldimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)-ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To the stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-formyl-2,5-dimethylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (˜0.748 mmol) in DCM from the previous reaction was added5-[(R)-2-amino-1-(tert-butyl-dimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-oneacetic acid salt (0.295 g, 0.748 mmol) in 1:1 MeOH:DCM (3.0 mL). Theresulting mixture was stirred at room temperature for 30 min and thensodium triacetoxyborohydride (0.476 g, 2.24 mmol) was added and stirringwas continued at room temperature for 4 h. Aqueous NaOH (1 M, 3 mL) andDCM (3 mL) were added and the layers were separated. The organic layerwas dried over sodium sulfate, filtered and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography(0-15% MeOH in DCM) to give the title compound (420 mg, 60% yield) as ayellow solid.

LC-MS (Method 10-90): Rt 2.39 min; m/z 931.6 [M⁺].

Example 47 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-{[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylEster Ditrifluoroacetic Acid Salt (Compound I-18)

A solution of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-{[(R)-2-(tert-butyl-dimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-2,5-dimethylphenylcarbamoyl)butyl]methylcarbamoyl}ethyl)piperidin-4-ylester (0.420 g, 0.451 mmol) and triethylamine trihydrofluoride (0.220mL, 1.35 mmol) in DCM (2.00 mL) was microwaved (300 watts) at 80° C. for10 min. The reaction mixture was concentrated under reduced pressure andthe residue was mixed with 1:1 AcOH:water and filtered. The filtrate waspurified by HPLC (Method 2-90) to give the title compound (124 mg, 26%yield) as a white solid.

LC-MS (Method 10-90): Rt 1.75 min; in/z 817.8 [M+H]⁺. ¹H NMR (CD₃OD) δ8.30 (m), 7.56 (m), 7.40 (m), 7.06 (m), 6.68 (d), 5.46 (m), 4.35 (s),3.51 (m), 3.30 (m), 3.16 (m), 2.94 (m), 2.42 (m), 2.27 (m), 1.97 (m),1.72 (m).

Example 48 Preparation of Methyl 3-Methylaminobenzoate

A solution of formic acid (106 mL) and acetic anhydride (53.0 mL) wasstirred at room temperature for 1 h. Methyl 3-aminobenzoate (8.50 g,56.2 mmol) was added and the resulting mixture was stirred overnight.The reaction mixture was then concentrated under reduced pressure togive a solid. The solid was dissolved in THF (50.0 mL) and this solutionwas cooled to 0° C. Borane dimethylsulfide (10.7 mL, 112 mmol) was addedslowly and the resulting mixture was stirred at room temperatureovernight. The reaction mixture was then cooled to 0° C. and MeOH (10mL) was added slowly. This mixture was stirred for 3 hours and thenconcentrated under reduced pressure to give the title compound, whichwas used without any further purification.

LC-MS (Method 10-90): Rt 1.25 min; m/z 166.4 [M+H]⁺.

Example 49 Preparation of 3-(Acryloylmethylamino)benzoic Acid MethylEster

To a stirred mixture of 3-methylaminobenzoic acid methyl ester (3.11 g,18.8 mmol) and sodium bicarbonate (3.16 g, 37.6 mmol) in DCM (20.0 mL)at 0° C. was added 2-propenoyl chloride (2.29 mL, 28.2 mmol). Thereaction mixture was stirred overnight at room temperature. Saturatedaqueous sodium bicarbonate solution was added and the layers wereseparated. The organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure to give the title compound (4.12 g,100% yield), which was used in the next reaction without furtherpurification.

Example 50 Preparation of3-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)benzoicAcid Methyl Ester

A stirred solution of 3-(acryloylmethylamino)benzoic acid methyl ester(estimated ˜56 mmol) and biphenyl-2-ylcarbamic acid piperidin-4-yl ester(16.6 g, 56.0 mmol) in EtOH (20.0 mL) was heated at 90° C. overnight.The reaction mixture was concentrated under reduced pressure and theresulting residue was purified by silica gel flash chromatography (0-10%MeOH in DCM) to give the title compound (13.0 g, 45% yield) as anoff-white solid.

LC-MS (Method 10-90): Rt 2.30 min; m/z 516.2 [M+H]⁺.

Example 51 Preparation of3-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)benzoicAcid

A solution of3-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionyl}methylamino)benzoicacid methyl ester (6.80 g, 13.2 mmol) and lithium hydroxide (1.58 g,65.9 mmol) in a 1:1 mixture of acetonitrile:water (25.0 mL) was stirredat room temperature overnight. The pH of the reaction mixture wasadjusted to pH 5 with aqueous hydrochloric acid (1M) and this mixturewas extracted with DCM (20 mL). The organic layer was dried over sodiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified by silica gel flash chromatography (0-10% MeOH in DCM) togive the title compound (5.80 g, 87% yield) as an off-white powder.

LC-MS (Method 10-90): Rt 2.15 min; m/z 502.2 [M+H]⁺.

Example 52 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(3-Chlorocarbonylphenyl)methyl-carbamoyl]ethyl}piperidin-4-ylEster

A mixture of3-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}-methylamino)benzoicacid (1.00 g, 2.00 mmol) and thionyl chloride (1.50 mL, 20.6 mmol) wasstirred at room temperature for 1 h. The reaction mixture was thenconcentrated under reduced pressure to give the title compound, whichwas used immediately in the next reaction without further purification.

Example 53 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-(1,3-Dioxolan-2-yl)-phenylcarbamoyl)phenyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of 4-(1,3-dioxolan-2-yl)phenylamine (0.0826 g,0.500 mmol) and N,N-diisopropylethylamine (348 μL, 2.00 mmol) in DCM(2.0 mL) at room temperature was added a solution ofbiphenyl-2-ylcarbamic acid1-{2-[(3-chlorocarbonylphenyl)methylcarbamoyl]ethyl}piperidin-4-yl ester(0.260 g, 0.500 mmol) in DCM (1.0 mL). The resulting mixture was stirredat room temperature for 1 h. LC-MS (Method 10-90) showed product waspresent (Rt 3.41 min; m/z 649.4 [M+H]⁺. Saturated aqueous sodiumbicarbonate solution was added and the layers were separated. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder reduced pressure. The residue was purified by silica gel flashchromatography (0-5% MeOH in DCM) to give the title compound (345 mgcontaining some solvent residue, ca. 100% yield) as a brown oil.

Example 54 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-Formylphenylcarbamoyl)-phenyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

To a stirred solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-(1,3-dioxolan-2-yl)phenylcarbamoyl)phenyl]methylcarbamoyl}ethyl)piperidin-4-ylester (345 mg containing some solvent residue, ca. 0.500 mmol) inacetonitrile (2.00 mL) at room temperature was added aqueoushydrochloric acid (1 N, 2.00 mL). The reaction mixture was stirred atroom temperature for 3 h. LC-MS (Method 10-90) showed product waspresent (Rt 3.45 min; m/z 605.0 [M+H]⁺. Saturated aqueous sodiumbicarbonate solution was added and the layers were separated. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder reduced pressure to give the title compound, which was used in thenext reaction without further purification.

Example 55 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-methyl}phenylcarbamoyl)phenyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

A solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-formylphenylcarbamoyl)-phenyl]methylcarbamoyl}ethyl)piperidin-4-ylester (151 mg, 0.250 mmol) and5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-oneacetic acid salt (98.6 mg, 0.250 mmol) in 1:1 MeOH:DCM (2.0 mL) wasstirred at room temperature for 3 h. Sodium triacetoxyborohydride (159mg, 0.750 mmol) was added and the reaction mixture was stirred at roomtemperature for 3 h. LC-MS (Method 10-90) showed product was present (Rt3.32 min; m/z 923.6 [M+H]⁺. Saturated aqueous sodium bicarbonatesolution was added and the layers were separated. The organic layer wasdried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound, which was used in the next reactionwithout further purification.

Example 56 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(4-{[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-3-methoxyphenyl)-methylcarbamoyl]ethyl}piperidin-4-yl EsterDitrifluoroacetic Acid (Compound I-25)

A solution of biphenyl-2-ylcarbamic acid1-{2-[(4-{[(R)-2-(tert-butyldimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-3-methoxyphenyl)methylcarbamoyl]ethyl}piperidin-4-ylester (231 mg, 0.250 mmol) and triethylamine trihydrofluoride (204 μL,1.25 mmol) in DCM (2.00 mL) was stirred at room temperature overnight.LC-MS (Method 10-90) showed product was present (Rt 2.73 min; m/z 809.6[M+H]⁺. The reaction mixture was concentrated under reduced pressure andthe residue was purified by HPLC (Method 10-50) to give the titlecompound (47.3 mg, 23.4% yield, 99% purity) as a white solid.

Example 57 Preparation of 4-Nitrobenzenesulfonic Acid2-(4-Nitrophenyl)ethyl Ester

To a stirred solution of 4-nitrobenzeneethanol (3.34 g, 20.0 mmol) andtriethylenediamine (3.36 g, 30.0 mmol) in DCM (62.7 mL) at 0° C. wasadded 4-nitrobenzenesulfonyl chloride (4.97 g, 22.43 mmol) in portionsover a 5 min period. The reaction mixture was then stirred at roomtemperature for 2 h. Water (50 mL) was added and the resulting mixturewas stirred for 10 min. The organic layer was separated, dried oversodium sulfate, filtered and concentrated under reduced pressure to givethe crude title compound (5.8 g, 82% yield) as a yellow solid.

HPLC (Method 2-90): Rt 4.23 min (214 nm).

Example 58 Preparation of5-[(R)-2-{Benzyl-[2-(4-nitrophenyl)ethyl]amino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

A stirred solution of 4-nitrobenzenesulfonic acid 2-(4-nitrophenyl)ethylester (3.78 g, 10.7 mmol);5-[(R)-2-benzylamino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(4.620 g, 8.975 mmol) and N,N-diisopropylethylamine (3.59 mL, 20.6 mmol)in acetonitrile (39.2 mL) was heated at 65° C. overnight. After coolingthe reaction mixture to room temperature, EtOAc (20 mL) was added andthis mixture was washed with brine (20 mL). The organic layer was driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was purified by silica gel chromatography (0-30% EtOAc inhexanes) to give the title compound (3.0 g, 50% yield) as a yellow oil.

LC-MS (Method 10-90): Rt 2.94 min; m/z 664.4 [M+H]⁺.

Example 59 Preparation of5-[(R)-2-{[2-(4-Aminophenyl)ethyl]benzylamino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

A mixture of5-[(R)-2-{benzyl-[2-(4-nitrophenyl)ethyl]amino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(3.00 g, 4.52 mmol); iron (2.52 g, 45.2 mmol) and iron(II) chloridetetrahydrate (0.18 g, 0.90 mmol) in EtOH (27.3 mL) and AcOH (9.09 mL)was stirred at 80° C. overnight. The reaction mixture was filteredthrough a pad of Celite, washing with EtOAc (20 mL). The organic layerwas washed with brine (10 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure to give the title compound, whichwas used without any further purification.

LC-MS (Method 10-90): Rt 2.43 min; m/z 634.4 [M+H]⁺.

Example 60 Preparation of Biphenyl-2-ylcarbamic Acid1-[2-({3-[4-(2-{Benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]-amino}ethyl)phenylcarbamoyl]phenyl}methylcarbamoyl)ethyl]piperidin-4-ylEster

A mixture of5-[(R)-2-{[2-(4-aminophenyl)ethyl]benzylamino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(1.27 g, 2.00 mmol);3-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)benzoicacid (1.00 g, 2.00 mmol); 1-hydroxy-7-azabenzotriazole (0.286 g, 2.10mmol) and N,N-diisopropylethylamine (0.697 mL, 4.00 mmol) in DCM (5.00mL) was stirred for 30 min at room temperature.N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.575 g,3.00 mmol) was added and the resulting mixture was stirred for 3 h.Saturated aqueous sodium bicarbonate solution (5 mL) was added and thismixture was extracted with DCM (2×5 mL). The combined organic layerswere dried over sodium sulfate, filtered and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography(0-10% MeOH in DCM) to give the title compound (1.74 g, 77% yield) as ayellow solid.

LC-MS (Method 10-90): Rt 2.73 min; m/z 1117.8 [M⁺].

Example 61 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-{2-[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-ethyl}phenylcarbamoyl)phenyl]methylcarbamoyl}ethyl)piperidin-4-ylEster

A solution of biphenyl-2-ylcarbamic acid1-[2-({3-[4-(2-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethyl]-amino}ethyl)phenylcarbamoyl]phenyl}methylcarbamoyl)ethyl]piperidin-4-ylester (1.74 g, 1.56 mmol) in EtOH (12.2 mL) and AcOH (0.122 mL) wasflushed with nitrogen for 3 min. Palladium on carbon (10%, ˜50% water,0.18 g) was added and the reaction mixture was flushed with hydrogen for4 min. The mixture was stirred under a hydrogen atmosphere (balloon)overnight and then filtered and concentrated under reduced pressure. Theresidue was purified by silica gel flash chromatography (0-15% MeOH inDCM) to give the title compound (1.11 g, 76% yield) as a thick brownishliquid.

LC-MS (Method 10-90): Rt 2.46 min; m/z 937.6 [M+H]⁺.

Example 62 Preparation of Biphenyl-2-ylcarbamic Acid1-(2-{[3-(4-{2-[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]ethyl}phenylcarbamoyl)-phenyl]methylcarbamoyl}ethyl)piperidin-4-ylEster Ditrifluoroacetate (Compound I-34)

A solution of biphenyl-2-ylcarbamic acid1-(2-{[3-(4-{2-[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-ethyl}phenylcarbamoyl)phenyl]methylcarbamoyl}ethyl)piperidin-4-ylester (estimated ˜1.56 mmol) and triethylamine trihydrofluoride (299 μL,1.84 mmol) in DCM (3.06 mL) was microwaved (300 watts) at 80° C. for 10min. The reaction mixture was concentrated under reduced pressure andthe residue was purified by HPLC (Method 10-50) to give the titlecompound (280 mg, 17%) as a white solid.

LC-MS (Method 10-90): Rt 1.98 min; m/z 823.4 [M+H]⁺.

Example 63 Preparation of [3-(2-Oxopropyl)phenyl]acetonitrile

A stirred solution of 3-bromophenylacetonitrile (10.0 g, 51.0 mmol),tributyltin methoxide (17.6 mL, 61.2 mmol),tris(dibenzylideneacetone)dipalladium(0) (500 mg, 0.5 mmol), isopropenylacetate (6.74 mL, 61.2 mmol) and2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl (800 mg, 2 mmol)in toluene (100 mL, degassed) was heated at 100° C. under nitrogen for 6h. The reaction mixture was cooled to room temperature and EtOAc (30 mL)was added. A solution of potassium fluoride (10 g, 200 mmol) in water(52 mL) was then added and the resulting mixture was stirred overnight.Brine was added and the mixture was filtered through a pad of Celite.The layers were separated and the organic layer was dried over sodiumsulfate, filtered (cotton plug) and concentrated under reduced pressure.The residue was purified by silica gel chromatography (0-30% EtOAc inhexanes) to give the title compound (6.1 g, 69% yield) as a brown oil.

¹H NMR (CD₃OD) δ 7.32 (t, 1H); 7.24 (d, 1H); 7.19 (s, 1H); 7.16 (d, 1H);3.85 (s, 2H); 3.77 (s, 2H); 2.14 (s, 3H).

Example 64 Preparation of{3-[(R)-2-((R)-1-Phenylethylamino)propyl]phenyl}acetonitrileHydrochloride Salt

To a stirred solution of [3-(2-oxopropyl)phenyl]acetonitrile (2.00 g,11.5 mmol) and (R)-1-phenylethylamine (1.52 mL, 11.9 mmol) in DCM (50.0mL) was added sodium triacetoxyborohydride (7.59 g, 35.8 mmol). Theresulting mixture was stirred at room temperature overnight. Aqueoussodium hydroxide (1 M, 10 mL) and saturated aqueous sodium bicarbonate(20 mL) were added and the layers were separated. The organic layer wasdried over sodium sulfate, filtered and concentrated under reducedpressure. The residue was partially purified by silica gelchromatography (0-5% MeOH in DCM). The resulting material was dissolvedin MeOH and acetyl chloride (0.5 mL) was added. This mixture wasconcentrated under reduced pressure and the residue was dissolved inMeOH (15 mL). Diisopropyl ether (30 mL) was added slowly to form asecond layer on top of the solution. After standing at room temperature,the titled compound (2.0 g, 55% yield) precipitated and was collected byfiltration.

¹H NMR (CD₃OD) δ 7.53-7.51 (m, 5H); 7.32 (t, 1H); 7.24 (t, 1H); 7.10 (s,1H); 7.05 (d, 1H); 4.64-4.59 (m, 1H); 3.21-3.16 (m, 2H); 1.70 (d, 3H);1.17 (d, 3H).

Example 65 Preparation of{(R)-2-[3-(2-Aminoethyl)phenyl]-1-methylethyl}-((R)-1-phenylethyl)amine

To a stirred solution of{3-[(R)-2-((R)-1-phenylethylamino)propyl]phenyl}-acetonitrilehydrochloride salt (2.00 g, 6.35 mmol) and cobalt(II) chloridehexahydrate (4.27 g, 18.0 mmol) in MeOH (40.0 mL) at 0° C. was addedsodium tetrahydroborate (2.72 g, 71.8 mmol) portion-wise (reaction wasexothermic). The reaction mixture was stirred for 1 h at roomtemperature and then concentrated aqueous hydrochloric acid was addedand stirring was continued until the solid that had formed wasbroken-up. The mixture was then made basic by addition of aqueous sodiumhydroxide (1 M). This mixture was filtered and the filtrate wasextracted with DCM (50 mL). The organic layer was dried over sodiumsulfate, filtered and concentrated under reduced pressure to give thetitle compound, which as used in the next reaction without furtherpurification.

LC-MS (Method 10-90): Rt 0.938 min; m/z 283.6 [M+H]⁺.

Alternatively, this reduction can be preformed using lithium aluminumhydride and cobalt(II) chloride hexahydrate in THF.

Example 66 Preparation of(2-{3-[(R)-2-((R)-1-Phenylethylamino)propyl]phenyl}ethyl)carbamic Acidtert-Butyl Ester

A solution of{(R)-2-[3-(2-aminoethyl)phenyl]-1-methylethyl}-((R)-1-phenylethyl)amine(ca. 6.35 mmol), di-tert-butyldicarbonate (1.41 g, 6.46 mmol) andN,N-diisopropylethylamine (1.50 mL, 8.62 mmol) in DCM (20.0 mL) wasstirred at room temperature for about 72 h. Saturated aqueous sodiumbicarbonate (20 mL) was added and the resulting mixture was extractedwith DCM (2×10 mL). The combined organic layers were dried over sodiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified by silica gel chromatography (0-5% MeOH in DCM) to give thetitle compound (1.24 g, 51% yield, 2 steps) as a colorless oil.

LC-MS (Method 10-90): Rt 2.42 min; m/z 383.2 [M+H]⁺.

Example 67 Preparation of {2-[3((R)-2-Aminopropyl)phenyl]ethyl}carbamicAcid tert-Butyl Ester

A stirred solution of(2-{3-[(R)-2-((R)-1-phenylethylamino)propyl]phenyl}ethyl)-carbamic acidtert-butyl ester (1.54 g, 4.02 mmol), palladium hydroxide on carbon (10wt. %, ˜50% water, 2.24 g, 0.805 mmol), and ammonium formate (1.27 g,20.1 mmol) in EtOH (50.0 mL) was heated gradually to 50° C. and thenstirred at 50° C. for 1.5 h. The reaction mixture was filtered through apad of Celite and the filtrate was concentrated under reduced pressureto give the title compound, which was used without further purification.

LC-MS (Method 10-90): Rt 1.91 min; m/z 279.4 [M+H]⁺.

Example 68 Preparation of[2-(3-{(R)-2-[(R)-2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)ethyl]carbamicAcid tert-Butyl Ester

A stirred solution of8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethyl-silanyloxy)ethyl]-1H-quinolin-2-one(3.14 g, 6.43 mmol); {2-[3-((R)-2-aminopropyl)-phenyl]ethyl}carbamicacid tert-butyl ester (1.79 g, 6.43 mmol) and triethylamine (1.08 mL,7.72 mmol) in DMF (21.0 mL) was microwaved (300 watts) at 80° C. for 15h. Saturated aqueous sodium bicarbonate (10 mL) was added and theresulting mixture was extracted with DCM (2×10 mL). The combined organiclayers were dried over sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by silica gel chromatography(0-5% MeOH in DCM) to give the title compound (2.8 g, 63% yield) as anoff-white solid.

LC-MS (Method 10-90): Rt 2.97 min; m/z 686.4 [M+H]⁺.

Example 69 Preparation of5-[(R)-2-{(R)-2-[3-(2-Aminoethyl)phenyl]-1-methylethylamino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

A solution of[2-(3-{(R)-2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)ethylamino]propyl}phenyl)ethyl]carbamicacid tert-butyl ester (2.8 g) in 20% trifluoroacetic acid in DCM wasstirred at room temperature for 3 h. Saturated aqueous sodiumbicarbonate solution was added to neutralize the TFA and the layers wereseparated. The organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure to give the title compound, whichwas used without further purification.

LC-MS (Method 10-90): Rt 1.91 min; m/z 586.4 [M+H]⁺.

Example 70 Preparation of Methyl(4-Formylaminophenyl)acetate

A solution of acetic anhydride (14.3 mL, 151 mmol) and formic acid (22.8mL, 605 mmol) was stirred at room temperature for 1 h.Methyl(4-aminophenyl)acetate (5.00 g, 30.3 mmol) was added and theresulting mixture was stirred overnight. Saturated aqueous sodiumbicarbonate (10 mL) was added and the resulting mixture was extractedwith DCM (2×10 mL). The combined organic layers were dried over sodiumsulfate, filtered and concentrated under reduced pressure to give thetitle compound (4.31 g) as a yellow oil, which was used in the nextreaction without further purification.

LC-MS (Method 10-90): Rt 1.57 min; m/z 194.4 [M+H]⁺.

Example 71 Preparation of Methyl(4-Methylaminophenyl)acetate

To a stirred solution of methyl(4-formylaminophenyl)acetate (4.31 g) inTHF (20.0 mL) at 0° C. was slowly added borane dimethylsulfide (8.61 mL,90.8 mmol). The reaction mixture was stirred at room temperatureovernight and then cooled to 0° C. MeOH (15 mL) was added cautiously andthe resulting mixture was stirred for 3 h. The mixture was thenconcentrated under reduced pressure to give the title compound (4.0 g,73% yield, 2 steps) as a clear oil.

LC-MS (Method 10-90): Rt 0.67 min; m/z 180.2 [M+H]⁺.

Example 72 Preparation of [4-(Acryloylmethylamino)phenyl]acetic AcidMethyl Ester

To a solution of methyl(4-methylaminophenyl)acetate (4.00 g, 22.3 mmol)and N,N-diisopropylethylamine (7.78 mL, 44.6 mmol) in DCM (20.0 mL) at0° C. was slowly added 2-propenoyl chloride (2.18 mL, 26.8 mmol). Thereaction mixture was stirred at room temperature for 2 h and thensaturated aqueous sodium bicarbonate (20.0 mL) was added. This mixturewas extracted with DCM (10 mL) and the organic layer was dried oversodium sulfate, filtered and concentrated under reduced pressure to givethe title compound, which was used in the next reaction without furtherpurification.

LC-MS (Method 10-90): Rt 2.20 min; m/z 234.0 [M+H]⁺.

Example 73 Preparation of[4-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}-methylamino)phenyl]aceticAcid Methyl Ester

A stirred solution of [4-(acryloylmethylamino)phenyl]acetic acid methylester (˜22.3 mmol) and biphenyl-2-ylcarbamic acid piperidin-4-yl ester(6.61 g, 22.3 mmol) in EtOH (15.0 mL) was heated at reflux overnight.The reaction mixture was concentrated under reduced pressure and theresidue was purified by silica gel chromatography (0-5% MeOH in DCM) togive the title compound (8.41 g, 71% yield, 2 steps) as a brown stickysolid.

LC-MS (Method 10-90): Rt 2.36 min; m/z 530.6 [M+H]⁺.

Example 74 Preparation of[4-({3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}-methylamino)phenyl]aceticAcid

A stirred solution of[4-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]-propionyl}methylamino)phenyl]aceticacid methyl ester (8.00 g, 15.1 mmol) and lithium hydroxide (1.81 g,75.5 mmol) in acetonitrile (20.0 mL) and water (20.0 mL) was heated at60° C. for 3 h. The pH of the reaction mixture was adjusted to pH 5 withaqueous hydrochloric acid (1 M) and the mixture was extracted with DCM.The organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography (0-20% MeOH in DCM) to give the title compound (6.7g, 86% yield) as an off-white solid.

LC-MS (Method 10-90): Rt 2.17 min; m/z 516.4 [M+H]⁺.

Example 75 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(4-{[2-(3-{(R)-2-[(R)-2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)-ethylamino]propyl}phenyl)ethylcarbamoyl]methyl}phenyl)-methylcarbamoyl]ethyl}piperidin-4-ylEster

A mixture of5-[(R)-2-{(R)-2-[3-(2-aminoethyl)phenyl]-1-methylethylamino}-1-(tert-butyldimethylsilanyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one(0.586 g, 1.00 mmol);[4-({3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionyl}methylamino)-phenyl]aceticacid (0.516 g, 1.00 mmol); 2-chloropyridium triflate on Wang resin(polymer-supported Mukaiyama reagent) (1.19 mmol/g loading; 1.68 g, 2.00mmol); and N,N-diisopropylethylamine (522 μL, 3.00 mmol) in DCM (5.00mL) was stirred at room temperature overnight. The reaction mixture wasfiltered and the filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (0-5% MeOH in DCM) togive the title compound (412 mg, 38% yield) as an off-white oil.

LC-MS (Method 10-90): Rt 2.65 min; m/z 1083.8 [M+H]⁺.

Example 76 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(4-{[2-(3-{(R)-2-[(R)-2-(tert-Butyl-dimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-propyl}phenyl)ethylcarbamoyl]methyl}-phenyl)methylcarbamoyl]ethyl}piperidin-4-ylEster

Dry nitrogen was bubbled into a stirred solution ofbiphenyl-2-ylcarbamic acid1-{2-[(4-{[2-(3-{(R)-2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethylsilanyloxy)-ethylamino]propyl}phenyl)ethylcarbamoyl]-methyl}phenyl)methylcarbamoyl]ethyl}-piperidin-4-ylester (0.704 g, 0.650 mmol) in EtOH (5.00 mL) for 3 min and thenpalladium on carbon (10 wt. %, ˜50% water; 0.289 g, 0.130 mmol) wasadded. Hydrogen was bubbled into the reaction mixture for 3 min and thenthe mixture was stirred under an atmosphere of hydrogen (balloon) for 1h. The reaction mixture was filtered and the filtrate was concentratedunder reduced pressure to give the title compound, which was usedwithout further purification in the next reaction.

LC-MS (Method 10-90): Rt 2.49 min; m/z 994.0[M+H]⁺.

Example 77 Preparation of Biphenyl-2-ylcarbamic Acid1-{2-[(4-{[2-(3-{(R)-2-[(R)-2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]propyl}phenyl)-ethylcarbamoyl]methyl}phenyl)methylcarbamoyl]ethyl}piperidin-4-ylEster Ditrifluoroacetic Acid Salt (Compound I-86)

A solution of biphenyl-2-ylcarbamic acid1-{2-[(4-{[2-(3-{(R)-2-[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]-propyl}phenyl)ethylcarbamoyl]methyl}phenyl)methylcarbamoyl]ethyl}piperidin-4-ylester (˜0.65 mmol) and triethylamine trihydrofluoride (184 μL, 1.13mmol) in DCM (3.00 mL) was microwaved (300 watts) at 80° C. for 10 min.The reaction mixture was concentrated under reduced pressure and theresidue was purified by HPLC (Method 10-50) to give the title compound(24.7 mg, 4% yield, 2 steps) as a white solid.

LC-MS (Method 10-90): Rt 1.78 min; in/z 879.8 [M+H]⁺.

By modifying the starting materials employed in the previous examples orby using similar procedures, the compounds shown in Table II wereprepared:

TABLE II Ex. ID Amount/ No. No. Compound LC-MS¹  78 I-2 

 39 mg Rt 2.94 min; m/z 789.6  79 I-3 

 14 mg Rt 2.90 min; m/z 805.8  80 I-4 

 9 mg Rt 3.18 min; m/z 805.6  81 I-5 

 50 mg Rt 2.20 min; m/z 803.6  82 I-6 

 20 mg Rt 2.15 min; m/z 839.6  83 I-8 

144 mg Rt 2.77 min; m/z 789.6  84 I-9 

 29 mg Rt 2.81 min; m/z 819.6  85 I-10

 38 mg Rt 2.93 min; m/z 803.6  86 I-11

 35 mg Rt 2.95 min; m/z 817.8  87 I-12

 10 mg Rt 2.23 min; m/z 817.6  88 I-15

 43 mg Rt 2.81 min; m/z 803.6  89 I-16

 10 mg Rt 2.18 min; m/z 819.8  90 I-17

 85 mg Rt 2.18 min; m/z 819.8  91 I-19

 30 mg Rt 3.52 min; m/z 853.4  92 I-20

 53 mg Rt 2.84 min; m/z 803.6  93 I-21

 46 mg Rt 2.82 min; m/z 803.6  94 I-22

 25 mg Rt 2.95 min; m/z 831.8  95 I-23

 10 mg Rt 2.26 min; m/z 831.6  96 I-24

 4 mg Rt 4.34 min; m/z 831.4  97 I-26

 21 mg Rt 2.78 min; m/z na  98 I-27

 92 mg Rt 2.66 min; m/z 823.6  99 I-28

 37 mg Rt 3.07 min; m/z 837.5 100 I-29

 28 mg Rt 2.76 min; m/z 839.4 101 I-30

 29 mg Rt 3.51 min; m/z 837.5 102 I-31

 45 mg Rt 3.42 min; m/z 873.6 103 I-32

 31 mg Rt 2.92 min; m/z 823.5 104 I-33

 48 mg Rt 3.21 min; m/z 837.5 105 I-34

 56 mg Rt 3.15 min; m/z 837.4 106 I-36

 20 mg Rt 3.16 min; m/z 837.4 107 I-37

 13 mg Rt 3.10 min; m/z 851.6 108 I-38

 5 mg Rt 4.15 min; m/z 837.5 109 I-39

 28 mg Rt 2.12 min; m/z 837.8 110 I-40

 7 mg Rt 3.06 min; m/z 837.5 111 I-41

 13 mg Rt 3.26 min; m/z 851.3 112 I-42

 5 mg Rt 3.64 min; m/z 837.6 113 I-43

 39 mg Rt 3.51 min; m/z 837.5 114 I-44

 11 mg Rt 3.20 min; m/z 851.5 115 I-45

 10 mg Rt 3.20 min; m/z 865.5 116 I-46

 6 mg Rt 3.11 min; m/z 851.6 117 I-47

 19 mg Rt 3.30 min; m/z 865.7 118 I-48

 41 mg Rt 3.47 min; m/z 851.5 119 I-49

108 mg Rt 3.58 min; m/z 851.6 120 I-50

 6 mg Rt 2.35 min; m/z 851.4 121 I-51

 6 mg Rt 2.33 min; m/z 851.4 122 I-52

 11 mg Rt 3.31 min; m/z 851.6 123 I-53

 11 mg Rt 3.17 min; m/z 851.4 124 I-54

 15 mg Rt 3.32 min; m/z 881.6 125 I-55

 15 mg Rt 2.33 min; m/z 865.6 126 I-56

 5 mg Rt 4.5 min; m/z 865.6 127 I-57

 7 mg Rt 4.38 min; m/z 865.4 128 I-58

 4 mg Rt 4.56 min; m/z 865.5 129 I-59

 65 mg Rt 2.02 min; m/z 823.6 130 I-60

 28 mg Rt 2.05 min; m/z 851.6 131 I-61

 41 mg Rt 2.32 min; m/z 887.6 132 I-62

 42 mg Rt 2.05 min; m/z 837.4 133 I-63

 34 mg Rt 2.02 min; m/z 837.4 134 I-64

na Rt 2.29 min; m/z 851.6 135 I-65

 5 mg Rt 2.33 min; m/z 851.6 136 I-66

na Rt 3.57 min; m/z 865.7 137 I-67

 15 mg Rt 3.70 min; m/z 865.4 138 I-68

 7 mg Rt 4.41 min; m/z 865.6 139 I-69

 7 mg Rt 4.50 min; m/z 856.6 140 I-70

 2 mg Rt 4.14 min; m/z 879.6 141 I-71

 5 mg Rt 2.37 min; m/z 879.6 142 I-72

 2 mg Rt 4.24 min; m/z 895.7 143 I-73

 3 mg Rt 4.14 min; m/z 883.6 144 I-74

 15 mg Rt 3.23 min; m/z 837.5 145 I-75

 10 mg Rt 3.63 min; m/z 837.5 146 I-76

 0.6 mg Rt 3.56 min; m/z 851.4 147 I-77

 15 mg Rt 4.31 min; m/z 865.3 148 I-78

 5 mg Rt 3.73 min; m/z 865.5 149 I-79

 10 mg Rt 3.50 min; m/z 865.5 150 I-80

 10 mg 151 I-81

 10 mg Rt 3.78 min; m/z 895.5 152 I-82

 10 mg Rt 3.78 min; m/z 895.6 153 I-83

 5 mg Rt 3.02 min; m/z 895.6 154 I-84

 15 mg Rt 3.50 min; m/z 883.6 155 I-85

 7 mg Rt 3.05 min; m/z 879.8 156 I-86

 5 mg Rt 4.45 min; m/z 879.6 157 I-88

 7 mg Rt 3.03 min; m/z 879.6 158 I-89

 8 mg Rt 4.53 min; m/z 879.5 159 I-90

 10 mg Rt 2.36 min; m/z 879.6 160 I-91

 20 mg Rt 3.61 min; m/z 879.6 161 I-92

 7 mg Rt 4.52 min; m/z 879.7 162 I-93

 4 mg Rt 4.24 min; m/z 879.7 163 I-94

 15 mg Rt 3.39 min; m/z 895.5 164 I-95

 12 mg Rt 4.10 min; m/z 883.5 165 I-96

 3 mg Rt 4.34 min; m/z 851.4 ¹Amount isolated (crude or pure) inmilligrams; Rt = retention time in minutes; LC-MS: observed m/z,typically [M + H]⁺; na = not available.Biological Assays and Preparations

Example A Cell Culture and Membrane Preparation from Cells ExpressingHuman M₁, M₂, M₃ and M₄ Muscarinic Receptors

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in Hams F-12 media supplemented with 10% FBS and 250 μg/mLGeneticin. The cells were grown in a 5% CO₂, 37° C. incubator and liftedwith 2 mM EDTA in dPBS. Cells were collected by 5 minute centrifugationat 650×g, and cell pellets were either stored frozen at −80° C. ormembranes were prepared immediately for use.

For membrane preparation, cell pellets were resuspended in lysis bufferand homogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG;20 seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for15 minutes at 4° C. The membrane pellet was then resuspended withre-suspension buffer and homogenized again with the Polytron tissuedisrupter.

The protein concentration of the membrane suspension was determined bythe method described in Lowry et al., 1951, Journal of Biochemistry,193, 265. All membranes were stored frozen in aliquots at −80° C. orused immediately.

Aliquots of prepared hM₅ receptor membranes were purchased fromPerkinElmer, Inc. (Wellesley, Mass.) and were stored at −80° C. untiluse.

Example B Radioligand Binding Assay for Muscarinic Receptors

Radioligand binding assays for cloned muscarinic receptors wereperformed in 96-well microtiter plates in a total assay volume of 100μL. CHO cell membranes stably expressing either the hM₁, hM₂, hM₃, hM₄or hM₅ muscarinic subtype were diluted in assay buffer to the followingspecific target protein concentrations (μg/well): 10 μg for hM₁, 10-15μg for hM₂, 10-20 μg for hM₃, 10-20 μg for hM₄, and 10-12 μg for hM₅ toget similar signals (cpm). The membranes were briefly homogenized usinga Polytron tissue disruptor (10 seconds) prior to assay plate addition.

Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM.

Displacement assays for determination of K_(i) values of test compoundswere performed with [³H]-NMS at 1 nM and eleven different test compoundconcentrations. The test compounds were initially dissolved to aconcentration of 400 μM in dilution buffer and then serially diluted 5×with dilution buffer to final concentrations ranging from 10 pM to 100μM. The order of addition and volumes added to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 6 hours at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer, Inc.) pre-treated in 1% BSA. Filter plateswere rinsed three times with wash buffer (10 mM HEPES) to remove unboundradioactivity. The plates were then air-dried and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer, Inc.) were added to each well.The plates were then counted in a PerkinElmer Topcount liquidscintillation counter (PerkinElmer, Inc.).

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the one-site competition model. K_(i) values for testcompounds were calculated from observed IC₅₀ values and the K_(D) valueof the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff WH. (1973) Biochemical Pharmacology, 22(23):3099-108). K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K; values for data reporting.

In this assay, a lower K_(i) value means the test compound has a higherbinding affinity for the receptor. hM₃ receptor binding (K_(i)) data forcompounds of this invention are shown in Table III.

Example C Cell Culture and Membrane Preparation from Cells ExpressingHuman β₁, β₂ or β₃ Adrenergic Receptors

Human embryonic kidney (HEK-293) cell lines stably expressing clonedhuman β1 and β₂ adrenergic receptors or Chinese hamster ovarian (CHO)cell lines stably expressing cloned human β₃ adrenergic receptors weregrown to near confluency in DMEM or Hams F-12 media with 10% FBS in thepresence of 500 μg/mL Geneticin. The cell monolayer was lifted with 2 mMEDTA in PBS. Cells were pelleted by centrifugation at 1,000 rpm, andcell pellets were either stored frozen at −80° C. or membranes wereprepared immediately for use.

For preparation of β₁ and β₂ receptor expressing membranes, cell pelletswere re-suspended in lysis buffer (10 mM HEPES/HCl, 10 mM EDTA, pH 7.4at 4° C.) and homogenized using a tight-fitting Dounce glass homogenizer(30 strokes) on ice.

For the more protease-sensitive β₃ receptor expressing membranes, cellpellets were homogenated in lysis buffer (10 mM Tris/HCl, pH 7.4)supplemented with one tablet of “Complete Protease Inhibitor CocktailTablets with 2 mM EDTA” per 50 mL buffer (Roche Molecular Biochemicals,Indianapolis, Ind.). The homogenate was centrifuged at 20,000×g, and theresulting pellet was washed once with lysis buffer by re-suspension andcentrifugation as described herein. The final pellet was thenre-suspended in ice-cold binding assay buffer (75 mM Tris/HCl pH 7.4,12.5 mM MgCl₂, 1 mM EDTA).

The protein concentration of the membrane suspension was determined bythe methods described in Lowry et al., 1951, Journal of BiologicalChemistry, 193, 265; and Bradford, Analytical Biochemistry, 1976, 72,248-54. All membranes were stored frozen in aliquots at −80° C. or usedimmediately.

Example D Assay for Determining Adrenergic Receptor Agonist Potency

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturers instructions. For this assay, HEK-293 cell linesstably expressing cloned human β₁ or β₂ receptors were grown to nearconfluency in DMEM supplemented with 10% FBS and Geneticin (500 μg/mL);or CHO-K1 cell lines stably expressing cloned human β₃ adrenergicreceptors were grown to near confluency in Hams F-12 media supplementedwith 10% FBS and Geneticin (250 μg/mL). Cells were rinsed with PBS anddetached in dPBS (Dulbecco's Phosphate Buffered Saline, without CaCl₂and MgCl₂) containing 2 mM EDTA or Trypsin-EDTA solution (0.05%trypsin/0.53 mM EDTA). After counting cells in Coulter cell counter,cells were pelleted by centrifugation at 1,000 rpm and re-suspended instimulation buffer containing TBMX (PerkinElmer Kit) pre-warmed to roomtemperature to a concentration of 1.6×10⁶ to 2.8×10⁶ cells/mL. About40,000 to 80,000 cells per well were used in this assay. Test compounds(10 mM in DMSO) were diluted into PBS containing 0.1% BSA in BeckmanBiomek-2000 and tested at 11 different concentrations ranging from 100μM to 1 pM. Reactions were incubated for 10 min at 37° C. and stopped byadding 100 μL of cold detection buffer containing [¹²⁵I]-cAMP (NENSMP004, PerkinElmer Life Sciences, Boston, Mass.). The amount of cAMPproduced (pmol/well) was calculated based on the counts observed for thesamples and cAMP standards as described in the manufacturer's usermanual.

Data were analyzed by nonlinear regression analysis with the GraphPadPrism Software package (GraphPad Software, Inc., San Diego, Calif.) withthe sigmoidal equation. The Cheng-Prusoff equation (Cheng Y, and PrusoffW H., Biochemical Pharmacology, 1973, 22, 23, 3099-108) was used tocalculate the EC₅₀ values.

In this assay, a lower EC₅₀ value means the test compound has a higherfunctional activity at the receptor tested. hβ₂ efficacy (EC₅₀) data forcompounds of this invention are shown in Table III.

Example E Einthoven Assay

This assay measures the ability of a test compound to providebronchoprotection against methacholine (MCh)-induced bronchoconstrictionin a rat.

Male Sprague-Dawley rats (Harlan, Indianapolis, Ind.), weighing between200 g and 350 g, were used for all studies.

Test compound or vehicle (sterile deionized water) were dosed byinhalation (IH) over a 10 min period in a pie shaped inhalation chamber(R+S Molds, San Carlos, Calif.) using 5 mL of dosing solution. Rats wereexposed to an aerosol, which was generated from an LC Star Nebulizer SetModel 22F51 (PART Respiratory Equipment, Inc. Midlothian, Va.) driven byBioblend (5% CO2/95% atmospheric air) at a pressure of 22 psi. Rats weredosed with 100 μg of test compound unless otherwise indicated.

At predetermined time points, rats were anesthetized with anintraperitoneal (IP) injection of 120 mg/kg inactin (thiobutabarbital).A supplemental dose (40 mg/kg, IP) was administered if the animalresponded to physical stimuli (e.g. toe pinch). The surgical site wasshaved and a 1-2 cm midline incision of the ventral aspect of the neckwas made. The jugular vein was isolated and cannulated with asaline-filled polyethylene catheter (PE-50) to allow IV infusion of MCh.Trachea was dissected free and cannulated with a 14G needle (#NE-014,Small Parts, Miami Lakes, Fla.). After placement of the trachealcannula, each rat was ventilated using a respirator (Model 683, HarvardApparatus, Inc., MA) set at a stroke volume of 1 mL/100 g body weight(but not exceeding 2.5 mL volume) and a rate of 90 strokes per minute. AT-connector was placed along the respirator expiratory tubing to allowfor measurement of changes in ventilation pressure (VP) using a Biopactransducer that was connected to a Biopac (TSD 137C) pre-amplifier. Bodytemperature was maintained at 37° C. using a heating pad.

Changes in VP were recorded using the Acknowledge Data CollectionSoftware (Santa Barbara, Calif.). Baseline values were collected for atleast 2.5 min. Rats were then challenged with non-cumulative intravenous(IV) infusions of 40 and 80 μg/kg MCh. MCh was infused intravenously for2.5 minutes from a syringe pump (sp210iw, World Precision Instruments,Inc., Sarasota, Fla.) at a rate of 2 mL/kg/min, with a 2 minute intervalbetween the two doses of MCh. Changes in ventilation pressure (cm H₂O)in treated animals are expressed as % inhibition of MCh responserelative to control animals.

Other bronchoconstrictors, such as histamine and acetylcholine, can beused in place of MCh in this assay. Additionally, guinea pigs can beused instead of rats.

In this assay, a higher % inhibition of the MCh response indicates thatthe test compound provided a greater bronchoprotective effect.Inhibition greater than or equal to 30 percent at 24 h is indicative ofa long duration of action. Bronchoprotection data for compounds of thisinvention are shown in Table III.

TABLE III ID hM₃ hβ₂ Broncho. ID hM₃ hβ₂ Broncho. No. K_(i) (nM)¹ EC₅₀(nM)² at 24 h³ No. K_(i) (nM)¹ EC₅₀ (nM)² at 24 h³ I-1 0.1 3 nd I-2 0.11 No I-3 0.1 1 Yes I-4 0.1 2 Yes I-5 0.1 1 Yes I-6 0.1 1 Yes I-7 0.1 1Yes I-8 0.1 1 Yes I-9 0.1 6 nd I-10 0.1 1 Yes I-11 0.1 1 Yes I-12 0.1 1No I-13 0.1 1 Yes I-14 0.1 1 Yes I-15 0.1 1 Yes I-16 0.1 1 Yes I-17 0.11 Yes I-18 0.1 1 Yes I-19 0.1 1 Yes I-20 0.1 1 Yes I-21 0.1 1 Yes I-220.1 1 Yes I-23 0.1 1 Yes I-24 0.1 1 Yes I-25 0.1 1 Yes I-26 0.2 1 YesI-27 0.1 1 No I-28 0.2 1 No I-29 0.1 1 No I-30 0.2 1 No I-31 0.2 1 YesI-32 0.1 1 No I-33 0.2 1 No I-34 0.1 1 Yes I-35 0.2 1 Yes I-36 0.2 1 YesI-37 0.2 1 No I-38 0.2 1 No I-39 0.2 1 No I-40 0.1 1 Yes I-41 0.3 1 NoI-42 0.2 1 No I-43 0.1 1 Yes I-44 0.1 1 No I-45 0.2 1 No I-46 0.1 1 YesI-47 0.2 3 nd I-48 0.3 1 Yes I-49 0.1 1 Yes I-50 0.4 nd nd I-51 0.2 1 ndI-52 0.2 1 No I-53 0.1 1 No I-54 0.2 2 nd I-55 0.2 1 Yes I-56 0.2 1 NoI-57 0.1 1 nd I-58 0.2 1 nd I-59 0.1 3 nd I-60 0.1 3 nd I-61 0.1 1 YesI-62 0.1 1 Yes I-63 0.1 1 nd I-64 0.3 1 No I-65 0.1 1 Yes I-66 0.2 1 NoI-67 0.1 1 Yes I-68 0.1 1 Yes I-69 0.1 1 Yes I-70 0.3 1 nd I-71 0.1 1 ndI-72 0.4 4 nd I-73 0.2 1 nd I-74 0.3 2 nd I-75 0.2 2 nd I-76 0.3 1 ndI-77 0.1 1 Yes I-78 0.3 1 Yes I-79 0.1 1 No I-80 0.2 1 No I-81 0.1 1 YesI-82 0.1 1 Yes I-83 0.2 2 Yes I-84 0.1 1 nd I-85 0.2 2 Yes I-86 0.2 1Yes I-87 0.1 1 Yes I-88 0.1 1 Yes I-89 0.1 1 Yes I-90 0.1 1 nd I-91 0.21 Yes I-92 0.2 1 Yes I-93 0.8 11 nd I-94 0.1 1 Yes I-95 0.2 1 Yes I-960.1 3 nd nd = not determined. ¹hM₃ Muscarinic Receptor Binding (K_(i))(data rounded to nearest 0.1 nM). ²hβ₂ Adrenergic Receptor AgonistPotency (EC₅₀) (data rounded to nearest 1 nM), ³Bronchoprotective effectpresent at 24 h, e.g., ≧30% inhibition of the MCh response at 24 h inthe rat Einthoven assay (100 μg).

The data in Table III demonstrate that all compounds tested had an hM₃receptor binding (K_(i)) value in the range of from 0.1 nM to 0.8 nM.Moreover, all compounds tested had an hβ₂ efficacy (EC₅₀) value in therange of from 1 nM to 11 nM. Additionally, a majority of the compoundstested in the rat Einthoven Assay (using 100 μg of test compound)provided a significant bronchoprotective effect (≧30% inhibition ofMCh-induced bronchoconstriction) 24 hours after administration.

What is claimed is:
 1. Biphenyl-2-ylcarbamic acid1-(2-{[3-(4-{2-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]ethyl}phenylcarbamoyl)-phenyl]methylcarbamoyl}ethyl)piperidin-4-ylester of formula:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising (a) a compound of claim 1, a pharmaceuticallyacceptable salt thereof; and (b) a pharmaceutically acceptable carrier.3. A pharmaceutical composition comprising (a) a compound of claim 1, apharmaceutically acceptable salt thereof; (b) a steroidalanti-inflammatory agent or a pharmaceutically acceptable salt or solvatethereof; and (c) a pharmaceutically acceptable carrier.
 4. Thepharmaceutical composition of claim 3, wherein the steroidalanti-inflammatory agent is a corticosteroid or a pharmaceuticallyacceptable salt or solvate thereof.
 5. A method for treating a pulmonarydisorder selected from chronic obstructive pulmonary disease and asthmain a patient comprising administering to the patient a compound of claim1, a pharmaceutically acceptable salt thereof.
 6. A method for treatinga pulmonary disorder selected from chronic obstructive pulmonary diseaseand asthma in a patient comprising: (a) administering to the patient acompound of claim 1, or a pharmaceutically acceptable salt thereof; and(b) administering to the patient a steroidal anti-inflammatory agent, ora pharmaceutically acceptable salt or solvate thereof; wherein step (a)and (b) are conducted simultaneously or sequentially in any order. 7.The method of claim 5 or 6, wherein the pulmonary disorder is chronicobstructive pulmonary disease.
 8. The method of claim 5 or 6, whereinthe pulmonary disorder is asthma.
 9. A method for producingbronchodilation in a mammal comprising administering to the mammal abronchodilation-producing amount of a compound of claim 1, apharmaceutically acceptable salt thereof.
 10. The method of claim 9,wherein the mammal is a human.
 11. A compound of the formula: